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Effects of changes in inspired gas composition on ventilation and breathing pattern in awake and hibernating… McArthur, M. Dawn 1986

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EFFECTS OF CHANGES IN INSPIRED GAS COMPOSITION ON VENTILATION AND BREATHING PATTERN IN AWAKE AND HIBERNATING GROUND SQUIRRELS By M. DAWN MCARTHUR B.Sc. (Hons) U n i v e r s i t y o f B r i t i s h Columbia 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1986 (c) M. Dawn M c A r t h u r In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D E-6 ( 3 / 8 1 } ABSTRACT During entrance into hibernation i n mammals, continuous breathing i s converted to intermittent breathing i n conjunction with the reductions i n metabolic rate (MR) and body temperature (Tg). The intermittent breathing pattern i s characterized by long non-ventilatory periods (Tjjyp) , which are interrupted by bursts of several breaths (Cheyne-Stokes r e s p i r a t i o n , CSR) i n some species, or by only sing l e breaths i n other species. The nature of t h i s species difference i n intermittent breathing pattern has remained a puzzle, due mainly to the problems of measuring v e n t i l a t i o n accurately i n undisturbed hibernating animals. In t h i s study, I examined the v e n t i l a t o r y responses of two species of ground s q u i r r e l that display each of the two intermittent breathing patterns during hibernation. These experiments were designed to t e s t the hypothesis that the species dif f e r e n c e i n intermittent breathing pattern can be explained on the basis of a difference i n s e n s i t i v i t y to hypoxia and hypercapnia. When awake and euthermic at a T A of 22°C, both the golden-mantled ground s q u i r r e l (Spermophilus l a t e r a l i s ) and the Columbian ground s q u i r r e l (S. columbianus) breathe continuously. V e n t i l a t i o n and v e n t i l a t o r y responses were measured i n awake animals using whole body plethysmography. In response to a decrease i n the f r a c t i o n a l inspired 0 2 concentration ( F 1 0 2 ) b e l o w 15-10% both species increased v e n t i l a t i o n mainly v i a an increase i n breathing frequency ( f ) ; t i d a l volume (V T) was increased only when F I 0 2 was lowered to 5%. When the f r a c t i o n a l i n s p i r e d C0 2 concentration ( F j C 0 2 ) w a s increased, both species responded by increasing f and V T, so that minute v e n t i l a t i o n increased l i n e a r l y with F I C 0 2 . In S. columbianus, these response patterns were unaffected by acute cold exposure. During per i o d i c arousal from hibernation i n the same species hypoxic responses remained the same, but hypercapnic responses appeared to be s l i g h t l y elevated. Relative to non-fossorial, non-hibernating mammals, the two ground s q u i r r e l species showed comparable hypoxic responses, but blunted hypercapnic responses. These v e n t i l a t o r y responses are t y p i c a l of burrowing animals, such as these ground s q u i r r e l s . In hibernating animals, v e n t i l a t i o n was measured using a face-mask and pneumotachograph. S. l a t e r a l i s exhibited CSR, while S. columbianus showed only sing l e breaths. Both species had s i m i l a r o v e r a l l l e v e l s of v e n t i l a t i o n and continued to show s i m i l a r v e n t i l a t o r y responses to hypoxic and hypercapnic gas mixtures. Due to the decreases i n T B and MR and the increase i n hemoglobin-0 2 a f f i n i t y during hibernation, hypoxic s e n s i t i v i t y was n e g l i g i b l e i n both species. In contrast, hypercapnic responses, though reduced i n absolute terms during hibernation, were i n f a c t enhanced r e l a t i v e to r e s t i n g MR. In both S. l a t e r a l i s and S. columbianus, v e n t i l a t i o n was increased during hypercapnic exposure s o l e l y by a decrease i n Tj^yp. This suggests that there has been a s h i f t i n c e n tral integration of afferent information, since i n contrast to euthermia, i n hibernation the non-ventilatory period, not the breath, appears to be the major c o n t r o l l e d variable of the breathing pattern. These patterns of v e n t i l a t o r y response to hypoxia and hypercapnia suggest that i n euthermia changes i n P 0 2 play a predominant r o l e i n v e n t i l a t o r y control, whereas i n hibernation v e n t i l a t i o n i s controlled by changes i n Pco2 or pH. This was found to be true f o r both species, despite the presence of two d i s t i n c t intermittent breathing patterns. Thus the r e s u l t s do not support the hypothesis that t h i s difference i n breathing pattern during hibernation can be accounted f o r by a difference i n s e n s i t i v i t y to r e s p i r a t o r y gases. This study has shown that v e n t i l a t o r y control mechanisms are maintained i n the deeply hibernating animal. Only hypoxic s e n s i t i v i t y i s reduced during hibernation; hypercapnic s e n s i t i v i t y appears, i n fact, to be augmented, presumably enabling the animal to e f f e c t i v e l y control i t s v e n t i l a t i o n to meet the requirements f o r gas exchange and acid-base balance. i v TABLE OF CONTENTS page Abstract ... i i L i s t of Tables v i L i s t of Figures v i i Acknowledgements x Chapter One: General Introduction 1 Chapter Two: Ve n t i l a t o r y Responses of Awake Ground Squirrels Introduction . 16 Materials and Methods 22 Results 32 Discussion 53 Chapter Three: V e n t i l a t o r y Responses of Hibernating Ground Squirrels Introduction 67 Materials and Methods 70 Results. 77 Discussion 96 Chapter Four: General Discussion 109 L i t e r a t u r e Cited 122 Appendix 1 133 v LIST OF TABLES page Table 1. Resting v e n t i l a t o r y variables i n awake ground s q u i r r e l s 34 Table 2. E f f e c t s of a l t e r a t i o n of inspired gas composition on v e n t i l a t o r y timing variables i n awake ground s q u i r r e l s 45 Table 3. E f f e c t s of hyperoxic, hyperoxic/hypercapnic, and hypoxic/hypercapnic gas mixtures on f, V T, and V i n awake ground s q u i r r e l s 50 Table 4. Ve n t i l a t o r y and breathing pattern va r i a b l e s i n hibernating ground s q u i r r e l s 79 Table 5. E f f e c t s of hyperoxic, hyperoxic/hypercapnic, and hypoxic/hypercpanic gas mixtures on f, V T, and "V i n hibernating ground s q u i r r e l s 91 Table 6. E f f e c t s of a l t e r a t i o n of inspired gas composition on v e n t i l a t o r y timing variables i n hibernating ground s q u i r r e l s 93 Table 7. E f f e c t s of hyperoxic, hyperoxic/hypercapnic, and hypoxic/hypercapnic gas mixtures on breathing pattern var i a b l e s i n hibernating ground s q u i r r e l s 95 Table 8. V e n t i l a t o r y variables i n various species of hibernating animals 99 v i LIST OF FIGURES page Figure 1. Schematic diagram of whole body plethysinograph arrangement 2 6 o Figure 2. Representative r e s t i n g breathing traces recorded i n awake ground s q u i r r e l s . . . 33a Figure 3. E f f e c t of decreasing F I 0 2 on V, V T, and f i n awake ground s q u i r r e l s 37a Figure 4. E f f e c t of decreasing F I 0 2 on V, V T, and f i n an i n d i v i d u a l Columbian ground s q u i r r e l under three conditions 38$ * Figure 5. E f f e c t of decreasing F I 0 2 on V, V T, and f i n a l l Columbian ground s q u i r r e l s under three conditions 3 9a Figure 6. E f f e c t of increasing F I C 0 2 on V, V T, and f i n awake ground s q u i r r e l s 42a Figure 7. E f f e c t of increasing F I C 0 2 on V, V T, and f i n an i n d i v i d u a l Columbian ground s q u i r r e l under three conditions 4 3a Figure 8. E f f e c t of increasing F I C 0 2 on V, V T, and f i n a l l Columbian ground s q u i r r e l s under three conditions. 44a Figure 9. Bar p l o t of e f f e c t of changes i n F I 0 2 a n d FIC02' a l ° n e o r * n combination, on V i n awake ground s q u i r r e l s 51a. v n page Figure 10. Hey Plot showing hypoxic and hypercapnic v e n t i l a t o r y responses of awake ground s q u i r r e l s compared to those of the r a t 59a Figure 11. Schematic diagram of experimental arrangement for recording v e n t i l a t i o n i n hibernating ground s q u i r r e l s 72a Figure 12. Representative breathing records showing timing and breathing pattern variables of hibernating ground s q u i r r e l s 76a Figure 13. Representative records of r e s t i n g breathing pattern i n hibernating ground s q u i r r e l s 78a Figure 14. E f f e c t s of hypoxia and hypercapnia on breathing pattern during hibernation 82a Figure 15. E f f e c t of decreasing F I 0 2 on V, V T, and f i n hibernating ground s q u i r r e l s 830 Figure 16. E f f e c t of decreasing F I 0 2 o n breathing pattern v a r i a b l e s i n hibernating ground squirrels.....84a Figure 17. E f f e c t of increasing F I C 0 2 o n v» v i " a n d f i n hibernating ground s q u i r r e l s 86a Figure 18. E f f e c t of increasing F I C 0 2 on breathing pattern variables i n hibernating ground s q u i r r e l s 87a Figure 19. Bar p l o t of e f f e c t of changes i n F I 0 2 and FIC02 a l ° n e o r ^ n combination, on V i n hibernating ground s q u i r r e l s 92s v i i i page Figure 20. Relationship between hypoxic v e n t i l a t o r y responses and Hb0 2 d i s s o c i a t i o n curves 102fi Figure 21. Comparison of the v e n t i l a t o r y responses of awake and hibernating ground s q u i r r e l s to decreasing F J Q 2 a n ( * increasing F I C 0 2 I l i a Figure 22. Comparison of the r e l a t i v e hypoxic v e n t i l a t o r y responses of awake and hibernating ground s q u i r r e l s 112a Figure 23. Comparison of the r e l a t i v e hypercapnic v e n t i l a t o r y responses of awake and hibernating ground s q u i r r e l s 114b Figure 24. Changes i n breathing pattern during entrance, hibernation, and arousal i n the golden-mantled ground s q u i r r e l 118a i x ACKNOWLEDGEMENTS I am indebted to my supervisor, Dr. William K . Milsom, f o r h i s support, advice, encouragement, and endless patience throughout t h i s study. I also thank Dr. Wayne Vogl f o r supplying the golden-mantled ground s q u i r r e l s , as well as Tim V i t a l i s , Dennis Primmett, Peter Chan, Cheryl Webb, and others for helping to trap the Columbian ground s q u i r r e l s . I am gr a t e f u l to Mr. and Mrs. B. Rogers of Penticton, B.C. for allowing me to trap on t h e i r ranch. F i n a l l y , I thank A l l e n Handley, Don Brandys, and James Johnston for t h e i r assistance with the con t r o l l e d environment chamber. x CHAPTER ONE GENERAL INTRODUCTION Hibernation i s perhaps the most e f f i c i e n t way an animal may cope with the combined environmental stresses of extreme cold and s c a r c i t y of food. This i s e s p e c i a l l y true f o r small homeotherms. Due to t h e i r higher surface area-to-volume r a t i o , small animals lose more heat to the environment than do large animals. Consequently, they must produce more metabolic heat to maintain an euthermic T B (30-38°C i n mammals) when T A i s low. When t h e i r food supply i s l i m i t e d , i t becomes even more d i f f i c u l t f o r them to remain euthermic. By entering hibernation, the small mammal reduces i t s gradient for heat loss to the environment and eliminates i t s need fo r food, thereby increasing i t s s u r v i v a l time i n a cold, foodless environment by several f o l d (Morrison, 1960; Hudson, 1978). The simplest d e f i n i t i o n of hibernation comes from the L a t i n root "hiberna", meaning winter. I f translated d i r e c t l y , hibernation could apply to any organism that becomes dormant during the winter. Winter dormancy has been observed i n a l l classes of vertebrates, i n many invertebrates, and i n some plants (Lyman, 1982). Cl e a r l y , the type of dormancy i s not the same i n a l l of these cases, and so a more precise d e f i n i t i o n of hibernation i s generally applied to mammals (and b i r d s ) . 1 In mammals, the degree, or depth, of dormancy varies depending on the species and on the environmental conditions. For example, the T B of a very small rodent such as the pygmy mouse Baiomys t a y l o r i w i l l f a l l at any time i f the animal i s denied food, even i f T A i s high (Lyman, 1982). S i m i l a r l y , the Tg and MR of bats f a l l markedly whenever the animal stops moving, even momentarily (Lyman and C h a t f i e l d , 1955). In contrast, the golden hamster Mesocricetus auratus must be exposed to cold for several days or weeks before i t w i l l become dormant (Hudson, 1978), while even i n the cold, the woodchuck Marmota monax must be denied food before i t w i l l hibernate (Goodrich, 1973). In l i g h t of these differences, Lyman (1982) has proposed that the various types of mammalian (and avian) dormancy be placed into the following two major categories: torpor and deep hibernation. He defines torpor as a state of i n a c t i v i t y i n which Tg declines, but not usually below 15-20°C. This includes the d a i l y torpor of some birds, bats, and small rodents, as well as the seasonal torpor of bears (Lyman, 1982). Deep hibernation i s defined as a state of i n a c t i v i t y i n which Tg f a l l s to a point near ambient, often as low as 2-5°C. This r e f e r s only to a seasonal event, i n which the animal enters hibernation i n the f a l l , undergoes a l t e r n a t i n g bouts of hibernation and p e r i o d i c arousal throughout the winter, and then becomes f u l l y 2 ac t i v e again i n the spring (Lyman, 1982). Several early investigators speculated that hibernation i s simply an exaggerated form of sleep; some even suggested that hibernation might be entered through sleep (see H a l l , 1836; Rasmussen, 1916). More recently, i t has been proposed that sleep, torpor, and deep hibernation are c l o s e l y r e l a t e d and are, i n fact, homologous adaptations f o r energy conservation (Glotzbach and Hell e r , 1978; H e l l e r et a l . , 1978; H e l l e r , 1982). The proposed homology i s based l a r g e l y on studies of thermoregulatory control during sleep and hibernation, but i s supported by e l e c t r o p h y s i o l o g i c a l and behavioural evidence as well (Walker et a l . , 1977; Florant et a l . , 1978) . The proposal that sleep and hibernation are homologous a c t i v i t y states may have important implications f o r the study of v e n t i l a t o r y control during hibernation. Due to the d i f f i c u l t i e s of using deeply hibernating animals as experimental models, very l i t t l e i s known about the p r e v a i l i n g mechanisms of v e n t i l a t o r y c o n t r o l . In contrast, control of breathing i n sleep has been extensively studied. I f the homology holds true, then i t may be possible to apply the p r i n c i p l e s of v e n t i l a t o r y control i n sleep to the state of hibernation. In mammals, there are three fundamental states of cent r a l nervous arousal: wakefulness (W), non-rapid-eye-movement (NREM) sleep, and rapid-eye-movement (REM) 3 sleep (Remitters, 1981; S u l l i v a n , 1981) . These are defined by several p h y s i o l o g i c a l and e l e c t r o p h y s i o l o g i c a l c r i t e r i a , i n cluding muscle tone (by electromyogram, EMG) and electroencephalographic (EEG) characterization. Wakefulness corresponds to i n t a c t muscle tone and high-frequency, low voltage EEG a c t i v i t y . In NREM sleep, the muscle tone i s decreased, and the EEG switches to a low-frequency, high-voltage pattern. In humans and some other mammals, NREM sleep i s sub-divided into four stages of progressively deeper sleep, with stages 3 and 4 corresponding to slow-wave sleep (SWS). In REM sleep, the EEG a c t i v i t y i s s i m i l a r to that of W, but most muscle tone i s absent. Other hallmarks of REM sleep include phasic eye movements and EEG spikes i n the pontine-geniculate-occipital cortex (Remitters, 1981; S u l l i v a n , 1981) . Wakefulness i t s e l f has been shown to have a profound e f f e c t on v e n t i l a t i o n , as i s obvious i n i n d i v i d u a l s with sleep apnea syndrome (Guilleminault et a l . , 1976; Cherniack, 1981). When these patients are awake v e n t i l a t i o n i s normal, but when they f a l l asleep, v e n t i l a t i o n becomes arrhythmic and apneic periods are common. A r t e r i a l oxygen saturation (Sa 0 2) begins to f a l l , continuing u n t i l an arousal threshold i s reached; the patient then wakes up and a c t i v e control of v e n t i l a t i o n ensues ( P h i l l i p s o n , 1978; Remitters, 1981; S u l l i v a n , 1981). In NREM sleep, v e n t i l a t i o n i s t y p i c a l l y regular and, 4 o v e r a l l , minute v e n t i l a t i o n (V) i s decreased compared to quiet W ( P h i l l i p s o n , 1978; S u l l i v a n , 1981). Reductions i n oxygen consumption (V 0 2) and carbon dioxide production ( V C 0 2 ) of 10-20 % have been recorded i n NREM sleep, implying that the metabolic v e n t i l a t o r y drive w i l l also be decreased ( P h i l l i p s o n , 1978). In addition, the f a l l i n alve o l a r v e n t i l a t i o n (V A) does not p a r a l l e l the decline i n MR, as would be expected i n awake subjects, suggesting that the c h a r a c t e r i s t i c s of central respiratory control have been a l t e r e d ( P h i l l i p s o n , 1978; Remitters, 1981; Su l l i v a n , 1981). S e n s i t i v i t y to input from peripheral and cen t r a l receptor groups i s maintained i n NREM sleep, but i s reduced compared to W. P h i l l i p s o n et a l . , (1976) found that the l e v e l of C0 2 required to e l i c i t a v e n t i l a t o r y response i n dogs was higher i n NREM sleep than i n W. This suggests that there i s a sleep-related s h i f t i n the response threshold of the c e n t r a l chemoreceptors to a higher C0 2 p a r t i a l pressure (PQO2^' o r a l ° w e r P H ( P h i l l i p s o n et a l . , 1976). In keeping with the evidence f o r reduced C0 2 s e n s i t i v i t y , a sustained increase i n a r t e r i a l PQO2 ( P aC02^ * s usually seen i n experimental animals and humans i n NREM sleep ( P h i l l i p s o n , 1978; S u l l i v a n , 1981). Most evidence indicates that the v e n t i l a t o r y response to hypoxia also remains i n t a c t i n NREM sleep ( P h i l l i p s o n , 1978; Bowes et a l . , 1981; Neubauer et a l . , 1981), although there i s some 5 debate as to whether or not i t i s reduced compared to W. In REM sleep, breathing i s t y p i c a l l y rapid and highly e r r a t i c ( P h i l l i p s o n , 1978; Remitters, 1981; S u l l i v a n , 1981). Most studies report that v e n t i l a t o r y output (measured as phrenic nerve discharge) and e f f e c t i v e V are increased i n REM sleep compared to W and to NREM sleep ( P h i l l i p s o n e l al.,1976; P h i l l i p s o n , 1978; Su l l i v a n , 1981), though some disagree (Orem et a l . , 1977; Neubauer et a l . , 1981). Measurements of P a C 0 2 indicate that V A i s decreased i n REM sleep compared to NREM sleep, mainly due to the s h i f t to rapid, shallow breathing and increased dead space v e n t i l a t i o n ( P h i l l i p s o n , 1978). The i r r e g u l a r breathing pattern exhibited by animals i n REM sleep appears to r e s u l t from the influence of REM-specific c e n t r a l nervous a c t i v i t y on brainstem r e s p i r a t o r y centres (Netick et a l . , 1977; Orem, 1980; Su l l i v a n , 1981). Ve n t i l a t o r y s e n s i t i v i t y to hypercapnia i s retained i n REM sleep, but i s greatly reduced compared to both NREM sleep and W. S i m i l a r l y , the v e n t i l a t o r y response threshold and s e n s i t i v i t y to hypoxia are much lower i n REM sleep than i n ei t h e r NREM sleep or W ( P h i l l i p s o n , 1978; Bowes et a l . , 1981; Neubauer et a l . , 1981; S u l l i v a n , 1981; Hedemark and Kronenberg, 1982). In hibernation, MR f a l l s to 1/30-1/50 of euthermic l e v e l s (Wang, 1978; Malan, 1982), and breathing frequency 6 f a l l s from about 100 breaths/minute to 1-5 breaths/minute (Lyman, 1951; Biorck et a l . , 1956; Landau and Dawe, 1958; Malan et a l . , 1973)). Pembrey and P i t t s f i r s t documented periodicbreathing patterns i n several hibernating species i n 1899. These observations have been confirmed i n many subsequent studies involving such species as dormice (Pajunen. 1970, 1974), hamsters (Lyman, 1951; Kr i s t o f f e r s s o n and Soivio, 1966) , hedgehogs (Biorck et a l . , 1956; K r i s t o f f e r s s o n and Soivio, 1964, 1967; Tahti, 1975; Tahti and Soivio, 1975), marmots (Endres and Taylor, 1930; Goodrich, 1973; Malan et a l . , 1973), and ground s q u i r r e l s (Lyman, 1951; Landau and Dawe, 1958; Hammel et a l . , 1968; Steffen and Riedesel, 1982). Some workers found the intermittent breathing pattern to consist of short bursts of rapid v e n t i l a t i o n separated by long non-ventilatory (apneic) pauses (Cheyne-Stokes r e s p i r a t i o n , CSR)(Pembrey and P i t t s , 1899; Biorck et a l . , 1956; Kr i s t o f f e r s s o n and Soivio, 1964,1967; Hammel et a l . , 1968; Pajunen, 1970,1974; Tahti, 1975; Tahti and Soivio, 1975; Steffen and Riedesel, 1982). Others noted that shorter non-ventilatory periods were interrupted by only one or two deep breaths (Endres and Taylor, 1930; Landau and Dawe, 1958; Malan et a l . , 1973). I t i s not known how these intermittent breathing patterns develop during entrance into hibernation, nor how they are co n t r o l l e d once the animal i s hibernating deeply. 7 Furthermore, there i s no evidence to explain why some species take s i n g l e breaths while others show CSR. Cheyne-Stokes r e s p i r a t i o n c l a s s i c a l l y occurs i n human subjects with congestive heart f a i l u r e or c e n t r a l nervous dysfunction (Lange and Hecht, 1962; Dowell et a l . , 1971). I t i s a l s o observed, however, i n sleeping subjects at sea l e v e l and i n awake and sleeping subjects acutely exposed to high a l t i t u d e (Cherniack, 1981; Weil et a l . , 1982). In general, CSR appears to develop when one or more of the following occurs: an increase i n the c i r c u l a t o r y delay between lungs and chemoreceptors, a decrease i n body 0 2 and C0 2 stores (lung, blood, t i s s u e ) , and/or an increase i n the s e n s i t i v i t y of peripheral and central chemoreceptor groups (Guyton et a l . , 1957; Khoo et a l . , 1982; Longobardo et a l . , 1982; Cherniack and Longobardo, 1983). In p a r t i c u l a r , the peripheral chemoreceptors have been shown to play a major r o l e i n the development of CSR at high a l t i t u d e , e s p e c i a l l y during sleep (Weil et a l . , 1982; L a h i r i et a l . , 1983). During hibernation, c i r c u l a t i o n time i s probably prolonged due to the combined e f f e c t s of decreased heart rate, decreased cardiac output and increased blood v i s c o s i t y (Lyman and C h a t f i e l d , 1955). Thus the delay time between lungs and chemoreceptors w i l l be longer, which could contribute to the development of CSR. I t seems reasonable to assume, however, that the increased 8 c i r c u l a t o r y delay i s matched by the metabolic depression of the t i s s u e s . In addition, i t does not seem l i k e l y that there would be a mismatch i n some species but not i n others. Nor do body gas stores probably play a major r o l e i n the development of CSR during hibernation. P 0 2 and P C 0 2 values i n blood and i n t i s s u e gas pockets indicate that stores are not decreased, but rather may be increased. For example, Musacchia and Volkert (1971) reported an increase i n P a o 2 f r o m 64.9 t o r r i n euthermia to 87.7 t o r r i n hibernation. S i m i l a r l y , Tahti and Soivio (1975) measured a P a 02 o f 1 2 0 t o r r i n the hibernating hedgehog. Acid-base studies indicate that while P a c o 2 m a y b e d e c ^ e a s e d at low T B, the t o t a l C0 2 content and bicarbonate l e v e l of a r t e r i a l blood are increased (Stormont et a l . , 1939; Lyman and Hastings, 1951; Clausen, 1966; Kent and Peirce, 1967; Bartels et a l . , 1969; Musacchia and Volkert, 1971; Goodrich, 1973; Tahti and Soivio, 1975; Kreienbuhl et a l . , 1976). As well, there i s no s i g n i f i c a n t change i n lung volume i n bats during hibernation (Lechner, 1985). I t seems probable, therefore, that CSR during hibernation stems from some a l t e r a t i o n i n v e n t i l a t o r y s e n s i t i v i t y of chemoreceptor groups to r e s p i r a t o r y gases. Euthermic hibernators are well-known fo r t h e i r high tolerance to hypoxia and hypercapnia, r e f l e c t i n g t h e i r f o s s o r i a l l i f e s t y l e (Biorck et a l . , 1956; Hiestand et a l . , 9 1957; F a l e s c h i n i and Whitten, 1975). The depression of metabolic rate during hibernation would be expected to enhance t h i s tolerance and could lead to such intermittent breathing, or at l e a s t account f o r the long non-ventilatory periods displayed by hibernating animals. Furthermore, i t has been proposed that as T b and MR f a l l during entrance int o hibernation, v e n t i l a t o r y C0 2 elimination becomes uncoupled from metabolic C0 2 production, so that a r e s p i r a t o r y acidosis develops (Malan, 1978,1982; Snapp and H e l l e r , 1981). This implies that there i s an a l t e r a t i o n of c e n t r a l v e n t i l a t o r y control during entrance into hibernation, s i m i l a r to that which occurs at the onset of sleep. To date, studies of v e n t i l a t o r y control during hibernation have been l i m i t e d by the d i f f i c u l t i e s of measuring v e n t i l a t i o n i n deeply hibernating animals. Most conventional methods of t i d a l volume (V T) measurement impose some r e s t r a i n t on the subject, which could cause a hibernating animal to arouse. Hence, the majority of investigators have resorted to methods that quantify breathing frequency ( f ) , but give only an i n d i c a t i o n of the depth of v e n t i l a t i o n . Only three groups have made quantitative measurements of V T during hibernation (Endres and Taylor, 1930; Malan et a l . , 1973; Steffen and Riedesel, 1982); v e n t i l a t o r y responses to hypercapnia were reported i n only one of these (Endres and Taylor, 1930). In contrast, several authors have recorded the e f f e c t s of hypoxia and hypercapnia on f and breathing pattern. In the 19th century, several workers compared the anoxia tolerance of hibernating animals with that of non-hibernators. For example, Spallanzani (1803) found that hibernating marmots would t o l e r a t e exposure to pure C0 2 f o r four hours; under these conditions, a r a t and a b i r d quickly died. S i m i l a r l y , H a l l (1836) observed that the hedgehog survived complete anoxia f o r only three minutes when awake, but f o r more than twenty minutes when hibernating. Biorck et a l . (1956) confirmed t h i s observation, reporting that a hibernating hedgehog could withstand exposure to pure N 2 for at l e a s t four hours, or twenty-eight times as long as a non-hibernating one. Tahti (1975) was the f i r s t to document the e f f e c t s of hypoxia and hypercapnia on breathing pattern i n a hibernating animal, the European hedgehog Erinaceus  Europaeus. CSR i s t y p i c a l of undisturbed v e n t i l a t i o n i n t h i s species; the non-ventilatory period (Tjjyp) between the bursts of v e n t i l a t i o n may be two to three hours long (Tahti, 1975; Tahti and Soivio. 1975). The hedgehog showed no v e n t i l a t o r y response to hypoxia u n t i l the i n s p i r e d l e v e l reached 16%, when a small decrease i n Tjjyp occurred, and o v e r a l l f was s l i g h t l y increased. As the i n s p i r e d F Q 2 was lowered, Tj^yp gradually became shorter u n t i l , at 3% 0 2 , breathing became continuous (Tahti, 1975). I f the hedgehog 11 was returned to a i r following exposure to 3% 0 2, CSR would reappear; however, i f exposure was prolonged, the animal would arouse from hibernation. The extreme tolerance of the hibernating animal to anoxia and hypoxia may simply be a consequence of i t s low MR and may not involve any a l t e r a t i o n of the s e n s i t i v i t y of the v e n t i l a t o r y control system to hypoxia during entrance into hibernation. The fa c t that a r t e r i a l P 0 2 f a l l s to very low l e v e l s ( i . e . 10 torr) during the non-ventilatory periods and the lack of breathing frequency response i n severe hypoxia has caused most previous investigators to conclude that changes i n 0 2 play l i t t l e r o l e i n v e n t i l a t o r y control during hibernation (Biorck et a l . , 1956; Tahti, 1975; Tahti et a l . , 1981; Steffen and Riedesel, 1982). In contrast, hibernating animals show pronounced v e n t i l a t o r y responses to hypercapnia. The reported threshold f o r an increase i n f ranges from l e s s than 1% FIC02 * n t h e hedgehog (Tahti, 1975) to 2-3% F I C 0 2 ^ n the golden hamster and t h i r t e e n - l i n e d ground s q u i r r e l (Lyman, 1951) and greater than 5% F I C 0 2 i n the marmot (Endres and Taylor, 1930). The o v e r a l l Cheyne-Stokes breathing pattern remains u n t i l the F I C 0 2 l e v e l i s raised above about 5%. Lyman (1951) found that breathing became continuous i n the hamster at 5-7% F I C 0 2 ; i n the hedgehog, i t becomes continuous i n some in d i v i d u a l s at 5-6% and i s 12 continuous i n a l l at 9% F I C 0 2 (Biorck et a l . , 1956; Tahti, 1975). As i n severe hypoxia, continuous breathing i n severe hypercapnia can be reconverted to CSR i f the animal i s returned to a i r ; otherwise, arousal from hibernation ultimately occurs (Tahti, 1975). Lyman (1951) compared the hypercapnic responses of hibernating hamsters and ground s q u i r r e l s to that of awake man and concluded that v e n t i l a t o r y s e n s i t i v i t y i s not depressed during hibernation. Conversely, Biorck et a l . (1956) d i r e c t l y compared the responses of hibernating and non-hibernating hedgehogs and found that the hypercapnic s e n s i t i v i t y was much lower i n hibernation. Tahti (1975) disagreed with these authors, s t a t i n g that the respiratory centres are not depressed during hibernation i n the hedgehog. In a l l of these studies, f was used as the major i n d i c a t i o n of v e n t i l a t o r y s e n s i t i v i t y . Since, i n non-hibernators, C0 2 i s known to have a strong e f f e c t on V T (Dejours, 1981), conclusions based on frequency changes alone may underestimate the t o t a l v e n t i l a t o r y response, and give the impression that v e n t i l a t o r y responses and s e n s i t i v i t y to hypercapnia are reduced i n hibernation when they are not. Endres and Taylor (193 0) are the only ones who have succeeded i n measuring both f and V T i n a hibernating animal during C0 2 exposure, concluding that the hypercapnic s e n s i t i v i t y of the marmot i s decreased during hibernation. 13 None of the availa b l e evidence o f f e r s any explanation f o r the apparent species difference i n intermittent breathing pattern. Based on the information presented here, t h i s study was designed to compare c e r t a i n aspects of v e n t i l a t o r y control i n species of ground s q u i r r e l that di s p l a y each of the d i f f e r e n t intermittent breathing patterns during hibernation. When awake and euthermic, both the Columbian ground s q u i r r e l (Spermophilus columbianus) and the golden-mantled ground s q u i r r e l (S. l a t e r a l i s ) breathe continuously. When hibernating, the Columbian ground s q u i r r e l takes only s i n g l e breaths, whereas the golden-mantled ground s q u i r r e l e xhibits CSR. The experiments were designed to t e s t the hypothesis that the changes i n breathing pattern that accompany entrance into hibernation r e s u l t from a change i n ve n t i l a t o r y s e n s i t i v i t y to respiratory gases. Further, i t was predicted that the difference i n intermittent breathing pattern would r e f l e c t a difference i n the s e n s i t i v i t y of the two species. To t h i s end, I have compared the e f f e c t s of changes i n the inspired gas composition on v e n t i l a t i o n and breathing pattern i n S. columbianus and S. l a t e r a l i s during both euthermia and hibernation. In the f i r s t section (Chapter 2) the v e n t i l a t o r y responses of awake animals of both species to changes i n the i n s p i r e d l e v e l s of 0 2 and C0 2 are examined. To compare t h e i r euthermic v e n t i l a t o r y responses, both species 14 were tested at a T A of 22°C. In addition, the responses of the Columbian ground s q u i r r e l were recorded during acute exposure to cold ( T A 5°C) and during p e r i o d i c arousal from hibernation ( T A 5°C) to assess the e f f e c t s of p r i o r hibernation on the v e n t i l a t o r y responses of the euthermic animal. In the second section (Chapter 3) the v e n t i l a t o r y responses of hibernating animals of both species are examined. Hibernating animals were exposed to the same l e v e l s of in s p i r e d 0 2 and C0 2 as awake animals, and the role s of hypoxia and hypercapnia i n the control of intermittent breathing during hibernation are discussed. In Chapter 4, the v e n t i l a t o r y responses of awake and hibernating ground s q u i r r e l s are compared and discussed. Such a comparison i s intended to address the question of whether or not v e n t i l a t o r y control i s depressed during hibernation, and also to o f f e r i n s i g h t into the difference i n intermittent breathing pattern displayed by these two species of ground s q u i r r e l during hibernation. 15 CHAPTER TWO VENTILATORY RESPONSES IN AWAKE GROUND SQUIRRELS INTRODUCTION Most animals that hibernate also l i v e i n underground burrows that protect them from predation and from the extremes of the surface climate. When the animal i s euthermic and active i n i t s burrow, a i r mixing and d i f f u s i v e exchange are not usually adequate to prevent the depletion of 0 2 and accumulation of C0 2 i n the burrow atmosphere (Vogel et a l . , 1973; Withers, 1978). The p r e v a i l i n g l e v e l s of hypoxia and hypercapnia depend on the number of burrow occupants and t h e i r metabolic rates, on burrow geometry, and on s o i l moisture and porosity (Withers, 1978; A r i e l i , 1979; Maclean, 1981). Reports of burrow gases i n the nests of non-hibernating and hibernating f o s s o r i a l animals are s i m i l a r . The f r a c t i o n a l 0 2 concentration ( F 0 2 ) i s t y p i c a l l y about 16%, but may be as low as 10%, and the f r a c t i o n a l C0 2 concentration ( F C 0 2 ) , i s t y p i c a l l y about 3%, but may be as high as 8-10% (Hayward, 1966; McNab, 1966; Scheck and Fleharty, 1971; Studier and Procter, 1971; Williams and Rausch, 1973; Maclean, 1981). Inhalation of such an hypoxic and hypercapnic atmosphere could lead to problems of 0 2 uptake and de l i v e r y to tissues as well as of C0 2 elimination and consequent acid-base perturbation. As such, burrowing animals have evolved a complex s u i t e of phy s i o l o g i c a l adaptations to compensate f o r the problems imposed by t h e i r l i f e s t y l e (Baudinette, 1974; Chapman and Bennett, 1975; Boggs et a l . , 1984). F o s s o r i a l and semi-fossorial mammals t y p i c a l l y have a higher 0 2 carrying capacity than do non-fossorial forms. Hematocrit (Hct), hemoglobin (Hb) concentration, and erythrocyte count a l l tend to f a l l at the upper end of the normal ranges f o r other mammals, though reports are va r i a b l e (Hall, 1965; Bartels et a l . , 1969; Baudinette, 1974; Harkness et a l . , 1974; Chapman and Bennett, 1975; Lechner, 1976; Ar et a l . , 1977). The 0 2 capacity i s further increased by a l e f t - s h i f t of the hemoglobin-oxygen ( H b 0 2 ) d i s s o c i a t i o n curve (Hall, 1965; Bartels et a l . , 1969; Baudinette, 1974; Lechner, 1976). The resultant 0 2 h a l f - s a t u r a t i o n pressures ( P 5 0 ) range from 23 t o r r i n the woodchuck, Marmota monax (Harkness et a l . , 1974) to 23-26 t o r r i n the ground s q u i r r e l s , Spermophilus  tereticaudus, S. beecheyi, and S. tridecemlineatus (Hall, 1965; Baudinette, 1974), and 33 t o r r i n the pocket gophers, Thomomvs bottae and T. umbrinus (Lechner, 1976). These are a l l lower than that of the rat , at 38-39 t o r r (Hall, 1965; Lechner, 1976). Such low P 5 0 values indicate high oxygen-binding a f f i n i t i e s that enable the burrower to saturate i t s a r t e r i a l blood even at the low 0 2 p a r t i a l pressures i t experiences i n the nest. 17 In addition, 0 2 unloading at tissues may be f a c i l i t a t e d i n f o s s o r i a l animals by the presence of an enhanced Bohr s h i f t (Ar et a l . , 1977; Boggs et a l . , 1984). C0 2 retention has the p o t e n t i a l e f f e c t of decreasing blood and t i s s u e pH and i n t e r f e r i n g not only with 0 2 binding v i a the Bohr e f f e c t (Dejours, 1981), but also with c e l l u l a r enzyme function (Malan, 1978). As an adaptation to minimize the e f f e c t s of chronic exposure to hypercapnia, f o s s o r i a l animals have higher blood and t i s s u e buffering c a p a c i t i e s than do t h e i r non-fossorial r e l a t i v e s . Both Chapman and Bennett (1974) and Lechner (1976) reported increased buffer l e v e l s i n pocket gophers compared to the white r a t . In conjunction with these adaptations for dealing with the l i m i t a t i o n s on gas exchange, burrowing animals are usually found to have a lower metabolic rate and r e s t i n g v e n t i l a t i o n than do non-burrowing animals. McNab (1966) measured basal metabolic rates (BMR) i n several f o s s o r i a l species that were 40 to 86% of those expected on the basis of body s i z e . In eight species of ground s q u i r r e l , Hudson and Deavers (1973) found that BMR was only about 60% of that predicted by standard allometric r e l a t i o n s h i p s ; BMR was described by the equation 3.24 W 0 , 6 6, where W i s mass. I t has been suggested that despite c h a r a c t e r i s t i c a l l y high Hb0 2 a f f i n i t y , hypoxia l i m i t s the a b i l i t y of the burrower to sustain a high metabolic rate. Baudinette 18 (1974) f e e l s that the r e l a t i v e l y lower BMRs of f o s s o r i a l mammals may be accounted f o r by the low P 5 0 values, and may represent an adaptation to avoid stress i n severe hypoxia. S e n s i t i v i t i e s to hypoxia and hypercapnia also appear to be reduced i n f o s s o r i a l mammals and birds (Soholt et a l . , 1973; F a l e s c h i n i and Whitten, 1975; A r i e l i and Ar, 1979; Boggs and Kilgore, 1983; Boggs et a l . , 1984; Holloway and Heath, 1984; Schlenker, 1985; Walker et a l . , 1985). V e n t i l a t o r y responses to hypercapnia have been given more attention than those to hypoxia, but i t i s c l e a r that f o s s o r i a l animals are highly t o l e r a n t of changes i n both. Euthermic golden-mantled ground s q u i r r e l s ( S . l a t e r a l i s ) show a smaller increase i n breathing frequency (f) during exposure to 8% inspired 0 2 than do the non-fossorial chipmunk, Tamias s t r i a t u s . and red s q u i r r e l , Tamiasciurus  hudsonicus. and can withstand complete anoxia f o r 4-8 times longer than can the other two species (Faleschini and Whitten, 1975). In the burrowing owl, v e n t i l a t i o n i s unchanged at 13% inspired 0 2, while i t i s increased by about 40% i n the non-burrowing bobwhite (Boggs and Kilgore, 1983). On the other hand, the golden hamster appears to have a s l i g h t l y greater v e n t i l a t o r y response to hypoxia compared to the white r a t (Holloway and Heath, 1984; Walker et a l . , 1985). S i m i l a r l y , the Djungarian hamster and the white mouse have the same hypoxic s e n s i t i v i t y (Schlenker, 19 1985). However, the response pattern d i f f e r e d between the species i n both of these studies; i t also d i f f e r e d between the two species of hamster (Schlenker, 1985; Walker et a l . , 1985) These r e s u l t s suggest that high hypoxic tolerance maynot be an universal phenomenon among the burrowers (Boggs et a l . , 1984) and that responses and s e n s i t i v i t y to hypoxia may d i f f e r even between c l o s e l y r e l a t e d species. In contrast, a high tolerance to hypercapnia i s t y p i c a l of almost a l l f o s s o r i a l mammals and b i r d s (Chapin, 1954; Soholt et a l . , 1973; Jahaveri et a l . , 1980; Boggs and Kilgore, 1983; Holloway and Heath, 1984; Schlenker, 1985; Walker et a l . , 1985). Both Holloway and Heath (1984) and Walker et a l . (1985) found a s u b s t a n t i a l l y lower hypercapnic response i n the golden hamster than i n the white r a t . Walker et a l . (1985) noted that during hypercapnic exposure the hamster was able to maintain a constant P a c o 2 ' w n i ^ e t n e r a t w a s not, despite i t s greater v e n t i l a t o r y response. A r i e l i and Ar (1979) also noted a blunted C0 2 s e n s i t i v i t y i n the kangaroo r a t . An exception may be the Djungarian hamster, which has a response s i m i l a r to that of the white mouse (Schlenker, 1985). As with hypoxic responses, therefore, i t i s possible that hypercapnic responses may vary between species, depending upon habitat and l i f e s t y l e . Whether or not there are species differences i n v e n t i l a t o r y responses among the burrowing hibernators i s 20 unknown, as very few studies have been conducted on hibernators i n the euthermic state. Given t h i s paucity of information and the range of species v a r i a b i l i t y , i t seemed possible that the difference i n intermittent breathing patterns displayed by hibernating Columbian and golden-mantled ground s q u i r r e l s might r e f l e c t a d i f f e r e n c e i n v e n t i l a t o r y s e n s i t i v i t y or v e n t i l a t o r y c o n t r o l . In the f i r s t part of the study, therefore, the v e n t i l a t o r y responses of awake, euthermic ground s q u i r r e l s of both species were examined at a T A of 22°C. In addition, i t was important to determine whether or not exposure to cold or the p h y s i o l o g i c a l changes concomitant with preparation f o r hibernation produced a s h i f t i n v e n t i l a t o r y c o n t r o l . Thus i n one species, S. columbianus. v e n t i l a t o r y responses were studied i n two a d d i t i o n a l conditions: i n euthermia at a T A of 5°C p r i o r to hibernation (late summer and early f a l l ) and i n euthermia during p e r i o d i c arousal from hibernation (late winter and e a r l y spring) also at T A 5°c. In t h i s chapter, the v e n t i l a t o r y responses of awake ground s q u i r r e l s to changes i n the l e v e l s of i n s p i r e d 0 2 and C0 2, alone or i n combination, under a l l four conditions w i l l be described. The responses of the two species w i l l be compared both to each other and with previous reports for other species. 21 MATERIALS AND METHODS Animals Ground s q u i r r e l s were used i n t h i s study because they are easy to obtain, to maintain i n c a p t i v i t y , and to induce int o hibernation. Adult Columbian ground s q u i r r e l s (Spermophilus columbianus) of both sexes were live-trapped on a ranch near Penticton, B r i t i s h Columbia i n l a t e June and early J u l y of 1983 and 1984. Adult female golden-mantled ground s q u i r r e l s (S. l a t e r a l i s ) were live-trapped near Redding, C a l i f o r n i a and transported to U.B.C. by a i r . In the laboratory a l l animals were housed I n d i v i d u a l l y i n p l a s t i c cages (46 x 24 x 15.5 cm or 46 x 24 x 21 cm) with mesh l i d s and given wood shavings f o r bedding. Purina lab chow and water were supplied ad  libitum; t h i s d i e t was supplemented with carrots or apples and sunflower seeds. The s q u i r r e l s were placed into a con t r o l l e d environment chamber at a of 20 + 1°C under 12L:12D photoperiod ( l i g h t s on 6AM). Animals used for study at T A s of 22°C (n=7) and 5°C (n=6) were maintained on t h i s regime. Periodic arousal animals (n=3) had been exposed to a T A of 5 + 1°C and a photoperiod of 2L:22D ( l i g h t s on 10AM) for at l e a s t two months, and had access to only lab chow and water. 22 Surgical procedures Electrodes f o r measurement of body temperature (T B) and heart rate (HR) were c h r o n i c a l l y implanted i n the ground s q u i r r e l s p r i o r to the summer experimental t r i a l s . T B was registered by a small thermistor, coated with epoxy, and positioned i n the abdominal cav i t y of the animal. A l l thermistors were c a l i b r a t e d f o r zero i n ice water before implantation. HR was recorded by means of three platinum leads implanted subcutaneously. The ground s q u i r r e l was anaesthetized with sodium pentobarbital (Somnotol, 30 mg/kg, i n t r a p e r i t o n e a l ) . The fur at the i n c i s i o n s i t e s was shaved away and the skin cleansed with a n t i s e p t i c (Bactine). Once deep reflexes had been abolished, a 1.5cm i n c i s i o n was made through the skin on the side of the abdomen. A small opening was made through the underlying body wall, the thermistor inserted into the body cavity, and the inner i n c i s i o n closed. Next, two of the platinum wires (one l i v e , one ground) were placed under the skin i n the lower abdomen and back regions r e s p e c t i v e l y . At t h i s point, a small i n c i s i o n was made through the skin at the nape of the neck. The connecting leads from the thermistor and HR leads were fed under the skin to the neckline opening and e x t e r i o r i z e d . The t h i r d platinum wire was positioned i n the scapular region v i a the neckline i n c i s i o n . A f t e r a l l implants had been checked for function, the outer i n c i s i o n s were closed and d i s i n f e c t e d . The s q u i r r e l was given Penbritin (30 mg/kg, intramuscular) post-operatively. The e x t e r i o r i z e d leads f o r the implants were anchored at the back of the neck. The thermistor s i g n a l was registered on a d i g i t a l - d i s p l a y monitor. The HR si g n a l was amplified (Gould DC preamplifier model 13-4615-10 or universal a m p l i f i e r model 13-4615-58) and displayed on a chart recorder (Gould 2400s). Measurement of v e n t i l a t i o n V e n t i l a t i o n was measured using the modified whole body plethysmograph technique of Jacky (1978,1980). The apparatus consisted of two rectangular 3.3 l i t r e chambers (14x23x12cm) made from 8mm t h i c k p l e x i g l a s s (Figure 1). One chamber, the "animal" chamber was rimmed with neoprene and the l i d f i t t e d with four latches f o r an a i r t i g h t s e a l . The other chamber, the "reference" chamber, was f u l l y sealed, but otherwise i d e n t i c a l to the animal chamber. Both had a i r inflow and outflow ports (0.75 cm diameter) i n e i t h e r end, as well as a port i n the l i d f o r the connection of transducer l i n e s , thermistor leads, etc. The subject was placed, unrestrained, into the animal chamber. Pressure f l u c t u a t i o n s within t h i s chamber concurrent with the warming and humidifying of inspire d a i r were compared to the pressure of the reference chamber, and measured by a d i f f e r e n t i a l pressure transducer (Validyne model DP103-18) connected between the two chambers. The o s c i l l a t i o n s i n 24 recorded pressure were considered to be proportional to t i d a l volume. Expired volume (V E) was calculated from the formula of Drorbaugh and Fenn (1955) as follows: pm x v c a l x T A< PB " PCH2C>) V E -P c a l [ T A ( P B - P C H 2 0 ) - T C ( P B - P ^ Q ) where Pm i s the observed pressure d e f l e c t i o n , Peal i s the c a l i b r a t i o n pressure d e f l e c t i o n , Vcal i s one-tenth the c a l i b r a t i o n volume, T A i s the body temperature of the animal (°K), T c i s the chamber temperature (°K), P B i s the barometric pressure, P A H 2 O * s t h e w a t e r vapour pressure at T A, and PCH20 * s w a t e r vapour pressure at T c (Jacky, 1978; Epstein et a l . , 1980). Water vapour pressure was not measured; chamber a i r was assumed to be saturated at T c. Calc u l a t i o n of V E by t h i s formula i s based on the assumption that expired gas returns from alveolar to ambient temperature and humidity before the next i n s p i r a t i o n occurs (Jacky, 1980). When t h i s i s not true, the Drorbaugh-Fenn formula tends to underestimate the true volume. Thus, Epstein and Epstein (1978) proposed a modification of the formula based on the assumption that expired gas must be considered to be at nasal, rather than Figure 1. Schematic diagram of whole body plethysmograph arrangement used to measure v e n t i l a t i o n i n awake ground s q u i r r e l s . See text f o r explanation. 26 N -CO to g a s a n a l y z e r s m a n o m e t e r ' r e f e r e n c e u n a n i m a l r e f r i g e r a t o r differential p r e s s u r e t r a n s d u c e r / \ T amplifier filter chart recorder o g a s a n a l y z e r s a l v e o l a r conditions. Jacky (1980) derived a factor to correct the Drorbaugh-Fenn V E value, such that V E / V C O R - 1 - T I / T T O T < 1 - G A / % ) where G A represents the pressure and temperature changes from a l v e o l a r to chamber conditions, and G N represents those from alveolar to nasal conditions. C a l i b r a t i o n of the system was performed dynamically with the animal present i n the chamber. A known volume of a i r was injected into the chamber at a known frequency, chosen to produce a pressure d e f l e c t i o n at l e a s t ten times as great as that of the animal's breathing (Jacky, 1978; Epstein et a l . , 1980). Records of v e n t i l a t i o n were taken with the system open. Preliminary studies showed that there was no a l t e r a t i o n of the pressure d e f l e c t i o n during eit h e r spontaneous breathing or c a l i b r a t i o n when the chamber was sealed. Nor was there any e f f e c t of ai r f l o w on the pressure d e f l e c t i o n s ; a t o t a l a i r f l o w rate of about 2 litres/minute was used i n a l l cases. Furthermore, measurements of V T by simultaneous pneumotachography and whole body plethysmography were i n close agreement. The advantage of using t h i s modified plethysmograph i s that the animal chamber i s purged constantly with fresh a i r so that 0 2 depletion and C0 2 accumulation are avoided. As well, the ins p i r e d gas composition can be altered r e a d i l y with minimal disturbance of the animal. Experimental protocol A l l experiments were done between 8AM and midnight. At the beginning of an experiment, the ground s q u i r r e l was weighed, placed into the animal chamber, and l e f t to adjust fo r about an hour; t h i s adjustment time was also necessary to s t a b i l i z e the animal chamber temperature. At the end of t h i s period, a r e s t i n g a i r control breathing trace was recorded. The animal was then exposed to the t e s t gas mixtures i n random order and a l t e r n a t i n g sequence with a i r controls. The duration of exposure to e i t h e r an a i r control or t e s t gas mixture was f i f t e e n minutes. The turnover time of chamber gases (2-3 minutes for a 3.3 l i t r e chamber at a t o t a l flow rate of 1-2 litres/minute) was taken into account and breathing traces f o r analysis were recorded during the l a s t minute of exposure. The t o t a l duration of an experimental t r i a l was approximately 8 hours. The t e s t gas mixtures were as follows: hypoxia, 15, 10, 5, or 3% f r a c t i o n a l i n s p i r e d 0 2 concentration ( F I 0 2 ) > hypercapnia, 2, 3, 5, or 7% f r a c t i o n a l i n s p i r e d C0 2 concentration ( F I C 0 2 ) * bypoxia/hypercapnia, 15 and 10% FI02 a t 3 a n c * 5 * FIC02' hyperoxia, 50% F I 0 2 ; hyperoxia/hypercapnia, 50% F I 0 2 at 3 and 5% F I C 0 2 (see Appendix I I ) . The t e s t gas mixtures were created by mixing room a i r with 100% C0 2, N 2, or 0 2 using flow meters, 28 and were analyzed to within + 0.1% with Beckman 0M11 0 2 and LB2 C0 2 analyzers. Both the inflow and outflow gas streams to the animal chamber were monitored continuously throughout the experiment. The t o t a l a i r f l o w of 2 litres/minute ensured that neither the F 0 2 nor the F C 0 2 was a l t e r e d by more than 0.5% by the animal's metabolism. The gas analyzers were c a l i b r a t e d d a i l y with room a i r and premixed 5 and 10% C0 2 (Radiometer GMA2 p r e c i s i o n gas supply). The animal chamber temperature ( T c ) , the animal's body temperature (T B) (where p o s s i b l e ) , and heart rate were recorded continuously throughout the t r i a l . Atmospheric pressure was noted at several times during the experiment; i t was r a r e l y found to d i f f e r over the time period. Nasal temperature (T N) was measured i n most animals at the end of the t r i a l using a small thermistor bead inserted approximately 0.5cm into the n o s t r i l . T N was found to be 32°C f o r a s q u i r r e l with a T B of 37°C, breathing about 60 times per minute at a T A of 22°C, and 29°C under s i m i l a r conditions i n animals at a T A of 5°C. These values were confirmed on a separate occasion i n 2 l i g h t l y anaesthetized s q u i r r e l s at both T A's, and agree with those previously reported (Schmid, 1976; Jacky, 1980; Fleming et a l . , 1983). The ground s q u i r r e l s were reweighed at the end of the t r i a l . They were not fasted p r i o r to study but usually there was no change i n mass from the beginning to the end of an experiment. Data analysis Approximately t h i r t y seconds of breathing trace was recorded at high speed (25 mm/second) at the end of each control or t e s t gas exposure. Breathing frequency (f) was counted as breaths per ten second i n t e r v a l and m u l t i p l i e d by s i x to give breaths per minute. Ten consecutive breaths were analyzed f o r the magnitude of the pressure d e f l e c t i o n (P-), t o t a l breath duration ( T T Q T ) ' i n s p i r a t o r y time (T j ) , expiratory time ( T E ) , and end-expiratory pause (T E') (see Figure 2). Expired volume was calcula t e d using the equations of Drorbaugh and Fenn (1954) as modified by Jacky (1978, 1980). The mean expired volume (V T) was mu l t i p l i e d by f to give minute v e n t i l a t i o n (V) i n ml/minute. Values of f, V T, and V for a l l control runs were averaged f o r each s q u i r r e l ; these mean values were then averaged to give a grand mean f o r each species. Calcul a t i o n of V 0 2 Data presented as a percent change from control (e.g. V/V 0 2) were calculated as the difference between the t e s t gas value and the mean control value f o r each i n d i v i d u a l . These transformed data were then averaged to give the grand mean percent change value f o r each species or state. For the c a l c u l a t i o n of V/V 0 2 i n Figures 10, 20, and 23, a value of 1.54 ml O2/100g/minute was used; t h i s was estimated from data on awake S. l a t e r a l i s (Heller, 1978) and S. r i c h a r d s o n i i (Wang, 1978) at T A's of about 20°C. 30 Despite the d i f f e r e n t sizes of S. columbianus (mean mass at T A 22°C / 485.7 +38.2 gm) and S. l a t e r a l i s (mean mass at T A 22°C, 221.5 + 11.7 gm), the data are not presented r e l a t i v e to body mass due to the d i f f i c u l t y of ascertaining the lean body mass of the i n d i v i d u a l . Seasonal changes i n body f a t content vary within as well as between species. Thus i t was f e l t that rather than making possible a c l e a r e r comparison between S. columbianus and S. l a t e r a l i s and between the 3 states i n S. columbianus. presentation of the v e n t i l a t o r y v a r i a b l e s and responses i n t h i s manner would be confusing. Hence absolute data are used i n most cases. Changes i n v e n t i l a t i o n within species were analysed by two-way analysis of variance, and Studentized Newman-Keul's multiple comparison t e s t s . Comparisons between species or conditions were made using unpaired t - t e s t s (Snedecor and Cochran, 1967; Zar, 1974). Differences were considered s i g n i f i c a n t at P<0.05, unless otherwise indicated. 31 RESULTS Resting v e n t i l a t i o n Figure 2 shows the r e s t i n g breathing patterns of awake Columbian and golden-mantled ground s q u i r r e l s . The corresponding v e n t i l a t o r y v ariables are given i n Table 1. A l l awake ground s q u i r r e l s breathed continuously, f was higher i n S. l a t e r a l i s (59.4 breaths/minute) than i n S. columbianus (48.6 breaths/minute) at T A 22°C, but V T was lower (0.54 vs. 0.63 ml), so that o v e r a l l V d i d not d i f f e r s i g n i f i c a n t l y between the two species. Expressed r e l a t i v e to body mass, V i s about twice as high i n the smaller species, S. l a t e r a l i s . T T 0 T , T j , and T E were also the same i n both species. Thus, duty cycle ( T j / T T 0 T ) was s i m i l a r f o r both, being, on average, 0.65 i n S. l a t e r a l i s and 0.60 i n S. columbianus. T E' was s l i g h t l y but not s i g n i f i c a n t l y longer i n S. columbianus. corresponding to the lower breathing frequency of t h i s species (Table 1). In S. columbianus, acute exposure to T A 5°C caused an increase i n mean V from 30.8 ml/minute to 35.9 ml/minute, due s o l e l y to a s i g n i f i c a n t increase i n f from 48.6 breaths/minute to 56.7 breaths/minute; V T was unchanged (Table 1). S i m i l a r l y , there was no change i n the timing of i n d i v i d u a l breaths, and the increased f r e f l e c t e d a decrease i n the duration of mean T E' from 0.96 seconds Figure 2. Representative r e s t i n g breathing traces recorded by whole body plethysmography i n both species of ground s q u i r r e l at a T A of 22°C, and i n the Columbian ground s q u i r r e l during acute cold exposure or p e r i o d i c arousal from hibernation. Awake animals breathed continuously under a l l conditions. The inse t shows the analysis of timing of each breath including i n s p i r a t o r y time (Tj), expiratory time ( T E ) , t o t a l breath duration ( T T O T ) ' a n d t n e end-expiratory pause ( T E ' ) . 33 G o l d e n - m a n t l e d ground squirrel awake 22 ° C Columbian ground squirre l awake 22 ° C a w a k e 5 0 C periodic a r o u s a l 5 0 C _ / Y _ . A _ A . A^J\, - . A - - A . . i 1 1 s e c 33a TABLE 1. Resting ventilatory variables in awake golden-mantled (S. lateralis) and Columbian (S. columbianus) ground squirrels. A l l values are •ean ±_ standard error. See text for explanation of symbols. S. la t e r a l i s S. columbianus S. columbianus S. columbianus awake 22°C awake 22°C awake 5°C per.arousal 5°C BOSS (g») T C TTOT (sec) 221.5 1 11.7 22.3 • 0.15 0.51 • 0.06 485.7 i 38.2 23.2 • 0.16 0.50 • 0.02 471.3 • 43.1 13.9 • 0.22 0.48 • 0.01 293.1 • 29.2 7.6 • 0.14 0.53 + 0.03 Ti (sec) 0.33 + 0.06 0.30 • 0.01 0.28 0.01 0.37 • 0.02 TE (sec) 0.17 0.01 0.20 0.01 0.20 + 0.02 0.17 • 0.01 TE' (sec 0.80 • 0.13 0.96 0.08 0.72 • 0.04 0.87 0.09 f (min) 59.4 • 3.5 48.6 • 2.0 56.7 • 2.4 50.9 • 3.0 VT (•1) 0.54 • 0.02 0.62 • 0.03 0.63 • 0.03 0.82 • 0.17 V (ml •in) 31.6 • 2.4 30.8 • 2.5 35.9 • 2.5 38.4 • 5.1 34 to 0.72 seconds. The T j / T T 0 T r a t i o remained the same as i n the 22°C animals, at 0.58 on average. In the three s q u i r r e l s tested, V was increased further to 38.4 ml/minute during p e r i o d i c arousal from hibernation. While t h i s represents a s i g n i f i c a n t l y higher V than that of animals at 22°C, i t i s not s i g n i f i c a n t l y d i f f e r e n t from that of animals acutely exposed to cold. An increase i n V T appears to contribute more to the r i s e i n V than does a change i n f. On the basis of these three animals, i t appears that breathing pattern i s also a l t e r e d during p e r i o d i c arousal. Although T T 0 T remained e s s e n t i a l l y the same as i n the other two conditions, T j was s l i g h t l y longer and T E somewhat shorter (Table 1). The change i n mean T E' followed the trend i n f; that of animals during per i o d i c arousal f e l l between those of the other two conditions, such that low f corresponded to high T E 1 and v i c e versa (Table 1). Responses to hypoxia The e f f e c t of decreasing the f r a c t i o n a l i n s p i r e d 0 2 concentration (F I 02) o n v e n t i l a t i o n i n S. l a t e r a l i s and S. columbianus awake at T A 22°C i s shown i n Figure 3. The two species showed remarkably s i m i l a r b r i s k v e n t i l a t o r y responses to hypoxia. There was no s i g n i f i c a n t d i f f e r e n c e of V, V T, or f between the two species at any l e v e l of F T O : > . In both, the V response threshold f e l l between 15 and 10% in s p i r e d 0 2. At 10% 0 2, V had increased by 117% i n S. l a t e r a l i s from the r e s t i n g value of 31.6 ml/minute to 60.4 ml/minute. In S. columbianus i t rose by 124%, from 30.8 ml/minute to 54.8 ml/minute. At 3% 0 2, V had increased by 463% to 152.8 ml/minute i n S. l a t e r a l i s and by 659% to 188.9 ml/minute i n S. columbianus. In both species the v e n t i l a t o r y response was achieved by increases i n f at a l l F I 0 2 l e v e l s ; V T was increased only i n severe hypoxia (3 and 5%) (Figure 3). At 3% 0 2, V T was s i g n i f i c a n t l y lower i n S. l a t e r a l i s compared to S. columbianus, but t h i s did not create any s i g n i f i c a n t d ifference i n V. This pattern of hypoxic v e n t i l a t o r y response i n S. columbianus was the same i n a l l three states, i . e . regardless of T A, season, or previous occurrence of hibernation (Figures 4 and 5). Figure 4 i l l u s t r a t e s the v e n t i l a t o r y responses of an i n d i v i d u a l ground s q u i r r e l tested under a l l three conditions. I t s responses were t y p i c a l of the groups. The mean values f o r a l l animals are shown i n Figure 5. The response threshold of 10-15% 0 2 was unaffected by cold. Despite the f a c t that V at 5% 0 2 d i d not d i f f e r s i g n i f i c a n t l y between T A s of 22 and 5°C, the acutely cold-exposed animals would not t o l e r a t e breathing 3% 0 2 f o r long enough to ascertain a steady-state response. Therefore these data are not included. Figure 3. E f f e c t of decreasing F j 0 2 on V, V T, and f i n awake Columbian ( o ) and golden-mantled ( A ) ground s q u i r r e l s . A l l values are mean + standard error f o r 5-7 animals. 37 250 37c, Figure 4. E f f e c t of decreasing F I 0 2 o n v * V T ' a n c * f i n an i n d i v i d u a l Columbian ground s q u i r r e l under three conditions: awake at T A 22°C ( O ) , awake at T A 5 ° c ( 0)/ a n d during p e r i o d i c arousal at T A 5°C 38 38o. Figure 5. E f f e c t of decreasing F I 0 2 on V, V T, and f i n the Columbian ground s q u i r r e l under three conditions: awake at T A 22 °C ( O ) (n=6), awake at T A 5°C (0 ) (n=6), and during p e r i o d i c arousal at T A 5°C ( 4 ) (n=3). A l l values are mean + standard error. 39 V (ml/min) Exposure to hypoxia caused a s i m i l a r decrease i n T T 0 T i n both species under a l l conditions, r e s u l t i n g from decreases i n both T j and T E (Table 2) . In S. l a t e r a l i s and S. columbianus at T A 22°C, T E' decreased as FI02 w a s l ° w e r e c ^ / u n t i l 3% 0*2, when i t increased but di d not return to control l e v e l s . In S. columbianus. both awake and during per i o d i c arousal at T A 5°C, T E' was decreased below control at a l l F I 0 2 l e v e l s (Table 2). In the l a t t e r state, T E' d i d not r i s e again at 3% 0 2, but instead continued to f a l l . Responses to hypercapnia The e f f e c t s of increasing F I C 0 2 o n v e n t i l a t i o n of awake ground s q u i r r e l s at T A 22°C are i l l u s t r a t e d i n Figure 6. As with the hypoxic v e n t i l a t o r y responses, the two species had s i m i l a r hypercapnic responses. In contrast, however, the hypercapnic response curves showed no c l e a r threshold. Between 0 and 7 % inspired C0 2, V rose s i g n i f i c a n t l y by 159% to 64.6 ml/minute i n S. l a t e r a l i s and by 166% to 89.3 ml/minute i n S. columbianus. U n t i l an F I C 0 2 of 5%, the pattern of response was the same i n both species: the increase i n V was due to s i m i l a r increases of both Vtp and f. But at 7% C0 2, the response patterns diverged, i n that f was increased further i n S_. l a t e r a l i s f whereas V T rose i n S. columbianus. This difference i n S. columbianus was not s i g n i f i c a n t , but f was s i g n i f i c a n t l y higher i n S . l a t e r a l i s at t h i s F I C 0 2 l e v e l . Acute cold exposure did not a l t e r the o v e r a l l hypercapnic responses of S . columbianus (Figures 7 and 8) or the pattern of v e n t i l a t o r y changes (Table 2). In contrast, the increase seen i n V was greater i n the p e r i o d i c arousal animals than i n e i t h e r of the other two conditions. For example, at 7% C0 2 V was 57.6 ml/minute i n S . columbianus at T A 5°C, compared to 116.3 ml/minute i n the three s q u i r r e l s during p e r i o d i c arousal from hibernation. The greater V response of p e r i o d i c arousal animals was due to a larger increase i n V T (Figure 8). In general, hypercapnic exposure had l i t t l e e f f e c t on v e n t i l a t o r y timing i n both S. l a t e r a l i s or S . columbianus (Table 2). At 7% C02, T T 0 T and T j were somewhat decreased i n S. l a t e r a l i s , r e f l e c t i n g the increase i n f; t h i s d i d not occur i n S . columbianus under any of the three conditions. In contrast to the hypoxic e f f e c t , T E was constant i n both species at a l l F I C 0 2 l e v e l s - The e f f e c t of hypercapnia on T E' was not consistent over the C0 2 range tested. Between 0 and 5% C0 2 there were v a r i a b l e changes i n mean T E', though at 7% C0 2 T E' was reduced below control i n both species (Table 2). This was true for S. columbianus under a l l conditions. 41 Figure 6. E f f e c t of increasing F I C 0 2 o n v / V T, and f i n awake Columbian (o) and golden-mantled ( A ) ground s q u i r r e l s at T A 22°C. A l l values are mean + standard error f o r 5-7 animals. 42 f (b reaths/min) V , (ml) Figure 7. E f f e c t of increasing F I C 0 2 on V, V T, and f i n an i n d i v i d u a l Columbian ground s q u i r r e l under three conditions: awake at T A 22°C ( O ) , awake at T A 5°C ( 0 ) , and during p e r i o d i c arousal at T A 5°C ( • ) . 43 I ml/min) Figure 8. E f f e c t of increasing Fico2 o n ^' V T ' a n d f i n the Columbian ground s q u i r r e l under three conditions: awake at T A 22°C ( o ) (n=6), awake at T A 5°C ( 0) (n=6), and during per i o d i c arousal at T A 5°C ( 4 ) (n=3). A l l values are mean + standard error. 44 44o TABLE 2. Effect of alteration of inspired gas composition on the ventilatory timing variables, total breath duration (TTOT* seconds), inspiratory time (Tj, seconds), expiratory time (TE, seconds), and end-expiratory pause (TE', seconds) in awake golden-mantled (S. lateralis) and Columbian (S. columbianus) ground squirrels. A l l values are mean standard error. GAS VARI- S.lateralis S.columbianus S.columbianus S.columbianus MIXTURE ABLE awake 22°C awake 22°C awake 5°C per.arousa!5C n 6 6 6 3 control TT0T 0.51 0.06 0.50 ••• 0.02 0.48 • 0.01 0.53 • 0.03 TI 0.33 0.06 0.30 0.01 0.28 • 0.01 0.37 • 0.02 TE 0.17 0.01 0.20 • 0.01 0.20 • 0.01 0.17 • 0.01 TE' 0.80 • 0.13 0.96 *• 0.08 0.72 • 0.04 0.87 0.09 15X02 TTOT 0.45 • 0.04 0.42 0.03 0.41 • 0.04 0.60 0.05 TI 0.27 0.05 0.22 • 0.03 0.22 0.04 0.42 • 0.06 TE 0.18 0.01 0.20 • 0.01 0.19 0.01 0.18 * 0.01 TE' 0.38 0.06 0.40 + 0.05 0.43 • 0.09 0.71 0.28 10*02 TTOT 0.45 • 0.07 0.37 • 0.02 0.36 • 0.01 0.31 0.03 TI 0.29 • 0.06 0.19 0.02 0.18 0.01 0.16 • 0.03 TE 0.16 • 0.02 0.18 • 0.01 0.18 • 0.01 0.15 • 0.003 TE' 0.37 • 0.09 0.33 + 0.05 0.36 0.07 0.49 • 0.09 5X02 TTOT 0.30 0.02 0.29 0.01 0.35 0.02 0.28 • 0.03 TI 0.15 • 0.01 0.13 0.01 0.15 • 0.01 0.14 • 0.03 TE 0.15 • 0.01 0.17 • 0.01 0.19 • 0.01 0.14 • 0.01 T E ' 0.14 0.03 0.18 • 0.05 0.24 • 0.05 0.26 • 0.09 3X02 TTOT 0.36 • 0.06 0.31 0.04 - 0.25 • 0.04 TI 0.21 • 0.05 0.14 • 0.04 0.13 • 0.02 TE 0.16 • 0.02 0.17 * 0.01 - 0.13 • 0.02 TE' 0.39 0.27 0.53 0.39 - 0.17 0.03 2XC02 TTOT 0.50 • 0.09 0.52 0.06 0.46 • 0.05 0.60 • 0.06 TI 0.33 0.06 0.31 0.04 0.25 • 0.04 0.43 0.07 TE 0.18 • 0.02 0.20 • 0.02 0.21 0.01 0.17 • 0.02 TE' 0.23 • 0.29 0.88 0.27 0.58 • 0.08 0.94 0.17 45 TABLE 2 continued... 3XCO2 TTOT TI TE TE' 5XCO2 TTOT TI TE T E ' 7XCO2 TTOT TI TE TE' 50XO2 TTOT TI TE T E ' 50XO2 TTOT 3XC02 T I TE TE' 15x02 TTOT 3XC02 Ti TE TE' 10x02 TTOT 3XC02 Ti TE TE' 50x02 TTOT 5XC02 Ti TE T E ' 15x02 TTOT 5XC02 Ti TE T E ' 10x02 TTOT 5XC02 Ti TE TE' 0.55 0.09 0.37 0.09 0.18 • 0.01 0.73 • 0.21 0.52 • 0.08 0.34 0.08 0.16 • 0.01 0.67 • 0.18 0.40 • 0.04 0.23 0.04 0.17 • 0.01 0.39 • 0.11 0.49 • 0.08 0.31 • 0.08 0.18 • 0.02 0.82 0.18 0.55 0.08 0.36 0.09 0.18 • 0.01 0.94 • 0.22 0.55 0.10 0.37 • 0.02 0.18 4 0.02 0.77 • 0.14 0.50 0.04 0.32 • 0.05 0.19 0.01 0.47 • 0.10 0.51 • 0.08 0.32 • 0.09 0.19 • 0.01 0.54 • 0.18 0.60 • 0.04 0.42 • 0.13 0.17 • 0.01 0.78 • 0.24 0.48 • 0.10 0.31 • 0.10 0.17 0.01 0.51 0.16 0.48 0.04 0.27 • 0.04 0.21 • 0.01 0.63 0.09 0.48 • 0.06 0.27 0.04 0.21 • 0.01 0.73 • 0.23 0.52 + 0.10 0.31 • 0.07 0.21 • 0.03 0.51 • 0.13 0.46 • 0.04 0.25 0.04 0.21 • 0.01 1.33 + 0.23 0.57 •* 0.04 0.37 • 0.05 0.20 • 0.01 1.12 • 0.25 0.45 0.03 0.25 • 0.03 0.21 • 0.01 0.57 + 0.08 0.39 • 0.03 0.21 • 0.02 0.19 • 0.02 0.38 • 0.07 0.50 0.09 0.31 + 0.08 0.21 0.01 1.00 4 0.35 0.39 • 0.03 0.28 • 0.03 0.18 + 0.01 0.67 + 0.23 0.43 • 0.03 0.23 4 0.02 0.20 • 0.02 0.45 0.06 0.50 0.05 0.29 • 0.05 0.21 + 0.01 0.60 • 0.08 0.46 • 0.03 0.25 • 0.03 0.21 • 0.01 0.43 • 0.07 0.55 • 0.06 0.33 0.06 0.22 0.01 0.66 • 0.27 0.60 • 0.08 0.40 0.07 0.20 • 0.01 1.27 • 0.16 0.61 4- 0.12 0.40 4- 0.11 0.21 • 0.01 1.25 • 0.46 0.50 • 0.05 0.30 * 0.04 0.20 * 0.01 0.58 4 0.16 0.47 4- 0.05 0.27 + 0.04 0.20 4- 0.01 0.32 4 0.06 0.55 4 0.07 0.35 4- 0.06 0.20 4 0.01 0.94 • 0.24 0.47 4- 0.05 0.39 4 0.06 0.21 4 0.01 0.86 4 0.18 0.52 4- 0.05 0.32 4- 0.05 0.20 • 0.01 0.38 • 0.07 0.53 • 0.04 0.36 • 0.04 0.17 • 0.003 0.68 • 0.19 0.46 i 0.07 0.29 i 0.06 0.16 1 0.003 0.35 • 0.10 0.57 • 0.09 0.40 4 0.09 0.17 4 0.01 0.50 • 0.34 0.35 4 0.01 0.18 4 0.03 0.18 4 0.02 0.56 4 0.14 0.60 4 0.09 0.42 • 0.09 0.18 4 0.01 1.20 • 0.43 0.61 4- 0.10 0.46 4 0.10 0.16 4 O.OO: 0.68 4 0.13 0.43 4 0.06 0.26 4- 0.06 0.17 4 0.01 0.25 4 0.03 0.62 • 0.19 0.44 4 0.18 0.18 • 0.01 0.75 4- 0.40 0.43 • 0.06 0.36 4 0.10 0.17 4 0.01 0.62 4 0.21 0.44 • 0.04 0.29 4 0.03 0.15 4 0.01 0.28 • 0.03 46 Responses to hyperoxia, hyperoxic hypercapnia, and hypoxic hypercapnia The e f f e c t s of hyperoxic, hyperoxic hypercapnic and hypoxic hypercapnic t e s t gas mixtures on v e n t i l a t i o n are given i n Tables 2 and 3. Figure 9 i s a bar p l o t of V values against i n s p i r e d gas composition f o r both species under a l l conditions. At T A 22°C, increasing F j 0 2 to 50% had no s i g n i f i c a n t e f f e c t on V i n ei t h e r species of ground s q u i r r e l . Hyperoxia tended to elevate V T i n both S. l a t e r a l i s and S. columbianus. but t h i s was o f f s e t by the decrease i n f, so that V remained e s s e n t i a l l y the same (Table 3). Although V T was increased over control i n both species, i t d i d not d i f f e r s i g n i f i c a n t l y between them. In S. columbianus. V T was increased from 0.62 ml to 0.75 ml and f decreased from 48.6 breaths/minute to 39.0 breaths/minute; t h i s resulted i n a decrease i n V to 29.4 ml/minute, or an average of 9.7% below c o n t r o l . In S. l a t e r a l i s . V T increased from 0.54 ml to 0.72 ml, and f rose s l i g h t l y from 59.4 breaths/minute to 62.0 breaths/minute; V was 46.2 ml/minute, an average increase of 6.7% above the control value. There was, however, a high v a r i a b i l i t y of i n d i v i d u a l responses to hyperoxia. This i s p a r t i c u l a r l y obvious i n S. l a t e r a l i s , where the mean V of 46.2 m/minute corresponds to values ranging from 13.2 to 144.4 ml/minute. In S. columbianus acutely exposed to T A 5°C, hyperoxia caused a decrease i n V s o l e l y through a decrease i n f, which f e l l from 56.7 breaths/minute to 34.2 breaths/minute, causing V to f a l l from 35.9 ml/minute to 21.2 ml/minute. There was no change i n V T (Table 3). During p e r i o d i c arousal, V was decreased i n hyperoxia, due to e f f e c t s on both f and V T. In these three s q u i r r e l s , f increased from 50.9 breaths/minute to 69.0 breaths/minute and V T decreased from 0.82 ml to 0.59 ml during hyperoxic exposure (Table 3). In both S. l a t e r a l i s and S. columbianus at T A 22°C, there was no s i g n i f i c a n t e f f e c t of hyperoxia on T T 0 T , T j , or T E; only T E' was increased s l i g h t l y (Table 2). In S. columbianus at T A 5°C hyperoxia caused an increase i n T T 0 T , T j , and T E. In contrast, these variables were a l l decreased i n S. columbianus during pe r i o d i c arousal (Table 2). In both S. l a t e r a l i s and S. columbianus at T A 22°C, the e f f e c t of changing F I 0 2 on the C0 2 response depended on the C0 2 l e v e l . The o v e r a l l V at 3% C0 2 was the same i n both species regardless of the concomitant F I 0 2 l e v e l (Table 3, Figure 9). As F I 0 2 was lowered, however, there tended to be a decrease i n the V T response and an increase i n the f response, suggesting a greater contribution of hypoxia to V (Table 3). At 5% C0 2, on the other hand, both hyperoxia and hypoxia augmented the V 48 response, mainly through a greater e f f e c t on V T (Table 3, Figure 9). In hypoxic hypercapnia, t h i s e f f e c t was observed at a higher F I 0 2 i n S. l a t e r a l i s (at 15%) than i n S. columbianus (at 10%). The same pattern of response was demonstrated by S. columbianus at T A 5°C at 3% C0 2; i n contrast, i n t h i s group, hyperoxia and hypoxia both decreased the V response at 5% C0 2, due mainly to a r e l a t i v e l y lower f response (Table 3). During per i o d i c arousal at T A 5°C, S. columbianus showed an augmentation of the V response at both C0 2 l e v e l s , though at 3% C0 2 only when F I 0 2 was lowered to 10% (Figure 9). This p o s i t i v e i n t e r a c t i o n at 3% C0 2 probably r e f l e c t s the r e l a t i v e l y greater response that was observed i n t h i s group i n normoxic hypercapnia (Figure 8 ) . The e f f e c t s of hyperoxic and hypoxic hypercapnia on v e n t i l a t o r y timing are included i n Table 2. Hyperoxia d i d not a l t e r the hypercapnia-induced changes i n v e n t i l a t o r y timing i n e i t h e r S. l a t e r a l i s or S. columbianus at T A 22°C: there was no s i g n i f i c a n t change i n T T 0 T , T j , or T E; T E' decreased as F I C 0 2 increased, but to the same degree i n normoxia and hyperoxia. Hyperoxia alone prolonged T E' i n S. columbianus. however, so that i t was always longer i n hyperoxic hypercapnia than i n normoxic hypercapnia (Table 2). In S. columbianus at T A 5°C, hyperoxia lead to increases i n T T 0 T and Tj at both TABLE 3. Effects of hyperoxic, hyperoxic/hypercapnic, and hypoxic/hypercapnic gas mixtures on breathing frequency (f, breaths/minute), t i d a l volume (Vj, ml), and minute ventilation (V, ml/minute) in awake golden-mantled (S. lateralis) and Columbian (S. columbianus) ground squirrels. A l l values given are mean standard error. GAS VARI- S.lateralis S.columbianus S.columbianus S.columbianus MIXTURE ABLE awake 22°C awake 22°C awake 5°C per.arousal 5C n 50X02 f OXCO2 VT V 50X02 f 3 X C O 2 VT v 15X02 f 3XC02 VT V 1 0 X 0 2 f 3XC02 VT V 50X02 f 5XC02 V T V 15X02 f 5XC02 VT V 1 0 X 0 2 f 5XC02 VT V 6 62.0 17.6 0.72 • 0.17 46.2 20.1 44.5 • 9.9 0.93 • 0.15 36.4 • 5.1 55.2 • 10.5 0.75 • 0.16 42.3 • 11.5 69.0 • 9.6 0.62 • 0.10 39.8 • 4.6 76.8 + 14.4 0.81 • 0.19 53.6 • 10.3 75.0 • 24.5 0.95 • 0.11 68.4 • 23.9 92.2 27.4 0.93 • 0.15 92.9 38.4 6 39.0 7.1 0.75 + 0.08 29.4 6.7 43.0 • 8.7 1.36 • 0.34 52.7 13.4 57.6 • 4.1 0.51 + 0.04 60.0 • 30.3 83.6 + 9.7 0.66 • 0.17 55.1 • 17.4 56.7 • 13.9 1.11 • 0.19 65.6 • 26.7 66.7 • 11.2 0.69 • 0.12 39.4 • 4.9 71.7 • 4.6 0.97 • 0.27 72.7 + 22.0 6 34.2 • 3.3 0.62 • 0.08 21.2 • 4.0 47.3 • 10.3 0.89 • 0.23 35.7 * 6.7 61.8 • 9.6 0.81 *• 0.08 50.4 • 7.8 60.4 9.0 0.66 • 0.11 52.8 • 10.7 44.1 • 6.9 0.96 • 0.15 30.7 • 3.6 43.1 6.9 0.75 • 0.06 30.7 • 3.6 70.3 • 7.9 0.74 • 0.09 50.6 * 6.2 3 69.0 • 10.6 0.59 * 0.08 39.5 • 4.7 41.5 • 15.4 0.96 • 0.10 40.8 1.74 46.7 • 7.3 0.87 4 0.23 39.0 4- 9.6 95.8 4- 6.8 1.25 • 0.32 122.4 * 38.3 47.5 l 13.9 1.27 • 0.22 83.1 1 14.6 62.5 • 21.4 1.49 • 0.28 83.1 • 14.6 85.0 i 5.0 1.04 • 0.21 111.3 • 26.4 50 Figure 9. Bar p l o t showing the e f f e c t of changes i n F I 0 2 and F j C 0 2 / alone or i n combination, on V of golden-mantled and Columbian ground s q u i r r e l s . The v e r t i c a l l i n e on each bar represents the standard error of the mean V. 51 Golden-mantled Columbian ground squirrel ground squirrel awake 22°C • awake 22°C • awake 5°C E3 periodic arousal 5°C 100-1 50 100-50 c £ c 100 50-100 50 J - X . 0 J I F - c o 2 , 0 / o 1 50 21 1 5 uT CN O 10 515, FIC02 l e v e l s , b u t there was no e f f e c t on T E; as at T A 22°C, T E' was increased by hyperoxia, but decreased with increasing F I C 0 2 (Table 2). During p e r i o d i c arousal i n S. columbianus, there were s l i g h t increases i n T T Q T and T j at both l e v e l s of hyperoxic hypercapnia, but no s i g n i f i c a n t decrease i n T E*, as was observed i n normoxic hypercapnia; T E did not change (Table 2). In S. l a t e r a l i s and S. columbianus, at T A 22°C, the changes i n v e n t i l a t o r y timing i n hypoxic hypercapnia followed the hypoxic pattern, i n that T T 0 T , T j , T E, and T E' a l l decreased as F I 0 2 w a s lowered (Table 2). The changes were usually l e s s severe i n hypoxic hypercapnia, however, as the decrease was le s s than that seen i n hypoxia alone, but greater than that of hypercapnia alone. This response pattern was also observed i n acutely cold-exposed S. columbianus at 3% C0 2, but not at 5% C0 2, when the changes i n v e n t i l a t o r y timing were les s than i n e i t h e r hypoxia or hypercapnia alone (Table 2). During p e r i o d i c arousal i n t h i s species, the changes i n TTOT a n d T I w e r e n o t a s great i n hypoxic hypercapnia as i n hypoxia alone, but the decrease i n T E' was augmented by the presence of C0 2 (Table 2). 52 DISCUSSION At T A 22°C, awake unrestrained golden-mantled and Columbian ground s q u i r r e l s breathe continuously and show s i m i l a r , strong v e n t i l a t o r y responses to hypoxia, but comparatively blunted responses to hypercapnia (Figures 2, 3, and 6). Acute exposure to a T A of 5°C does not a f f e c t the v e n t i l a t o r y responses of S. columbianus, while previous occurrence of hibernation may a l t e r some aspects of v e n t i l a t o r y control, p a r t i c u l a r l y breathing pattern and hypercapnic responses, i n t h i s species (Table 1, Figure 8). Whole body plethysmography has been used i n numerous studies to measure the v e n t i l a t i o n and v e n t i l a t o r y responses of several rodent species (Chapin, 1954; Pappenheimer, 1977; L a i et a l . , 1978; A r i e l i and Ar, 1979; Javaheri et a l . , 1980; Holloway and Heath, 1984; Chappell, 1985; Schlenker, 1985; Walker et a l . , 1985). The accuracy of V T c a l c u l a t i o n using t h i s technique depends on the p r e c i s i o n of the measurement of independent variables such as P B, Tg, and T c (Malan, 1973; Jacky 1978, 1980; Epstein et a l . , 1980). In addition, changes i n breathing pattern have been shown to a f f e c t the f i d e l i t y of the pressure measurements (Epstein et a l . , 1980; Jacky, 1980; Fleming et a l . , 1983). The V T values derived from whole body plethysmography f o r awake S. l a t e r a l i s and S. columbianus 53 are quite low compared to those expected on the basis of body s i z e (Stahl, 1967; Schmidt-Nielsen, 1984). For instance, Stahl (1967) predicts a V T of 1.0-2.7 ml for a 250gm r a t . The values I measured i n S. l a t e r a l i s and S. columbianus were 0.54 ml and 0.62 ml, resp e c t i v e l y (Table 1). Breathing frequency values f a l l within the range of 45-105 breaths/minute l i s t e d by Stahl (1967); however, the V values are only about 50% of that predicted (Table 1). The e f f e c t s of burrowing on V T are not clear, but o v e r a l l f and V are generally lower i n f o s s o r i a l and semi-fossorial species than i n non-fossorial ones (Boggs et a l . , 1984). The e f f e c t on V T may be species dependent, since Schlenker (1985) found a lower V T i n the Djungarian hamster than i n the mouse, but Walker et a l . (1985) reported a higher V T i n the Syrian hamster than i n the r a t . The r e l a t i v e l y low V T values recorded f o r S. l a t e r a l i s and S. columbianus may r e f l e c t some c h a r a c t e r i s t i c of the plethysmograph technique I employed, although the agreement of V T values measured simultaneously by pneumotachography and plethysmography argues that the discrepancy does not l i e i n the method. Furthermore, the measurements of were f a i r l y consistent from animal to animal and from condition to condition. In addition, the V T values obtained f o r d i f f e r e n t t e s t gases can s t i l l be compared since the system 54 and the c a l i b r a t i o n techniques were unchanged. Although the V l e v e l s of the two species of ground s q u i r r e l were s i m i l a r , f was somewhat higher and V T lower i n S . l a t e r a l i s than i n S. columbianus (Table 1). Mesurements of V and f showed considerable v a r i a b i l i t y among i n d i v i d u a l s q u i r r e l s , but t h i s i s not unexpected when awake, wholly unrestrained animals are studied. Relative to body mass, V was twice as high i n S. l a t e r a l i s . Thus i f the V T and V data were presented normalized to mass, a l l values f o r S. l a t e r a l i s would have been much higher than those f o r S . columbianus. The v e n t i l a t o r y responses recorded f o r both species would have remained r e l a t i v e l y the same, and so conclusions drawn from absolute responses are s t i l l v a l i d . In S. columbianus. r e s t i n g v e n t i l a t i o n was increased by both acute cold exposure and p e r i o d i c arousal from hibernation (Table 1). While V did not d i f f e r s i g n i f i c a n t l y between these two conditions, the increase was achieved i n d i f f e r e n t ways. In cold-exposed s q u i r r e l s , there was a r i s e i n f and no change i n V T, whereas during p e r i o d i c arousal, V T was increased with no change i n f. The greater f i n S . columbianus awake at T A 5°C implies the expected increase i n metabolic rate i n the cold, though t h i s was not measured. To my knowledge, no data e x i s t to allow d e f i n i t i o n of the thermoneutral zone i n S . columbianus, but T A 5°C (and T c 13°C) i s apparently 55 outside thermoneutrality, as i t i s i n the 13-lined ground s q u i r r e l , golden hamster, and deer mouse (Bullard and Meyer, 1966; Chappell, 1985). The r i s e i n V T during p e r i o d i c arousal at the same T A suggests an a l t e r a t i o n of v e n t i l a t o r y control, and probably r e f l e c t s the greater e f f e c t of C0 2 on v e n t i l a t i o n i n t h i s group (see below). Both S. l a t e r a l i s and S. columbianus exhibited strong v e n t i l a t o r y responses to decreased F I 0 2 (Figure 3); i n the l a t t e r species, the responses were not a l t e r e d by acute cold exposure or previous occurrence of hibernation (Figure 5). In a l l cases, the threshold f o r the hypoxic v e n t i l a t o r y response of both S. l a t e r a l i s and S. columbianus was about 10% 0 2, a f t e r which the response curves rose steeply (Figure 3). This threshold i s s l i g h t l y lower than that generally reported f o r non-fossorial mammals (Dejours, 1981; Dempsey and Forster, 1981; L a h i r i and Gelfand, 1981), but i s the same as that of the non-fossorial white r a t and porcupine, and the semi-fossorial woodchuck, echidna, and marmot (Leitner and Malan, 1973; Pappenheimer, 1977; Cragg and Drysdale, 1983; Boggs et a l . , 1984), and i s somewhat higher than that of the f u l l y f o s s o r i a l mole r a t ( A r i e l i and Ar, 1979). The o v e r a l l v e n t i l a t o r y responses of the two ground s q u i r r e l species were s i m i l a r not only to each other, but also to those found i n two other f o s s o r i a l hibernators, the Djungarian hamster (Schlenker, 1985) and the Syrian hamster (Holloway and Heath, 1984; Walker et a l . , 1985). At 10% in s p i r e d 0 2, V had increased by 117% i n S. l a t e r a l i s and by 124% i n S. columbianus. compared to 113% i n the Syrian hamster (Walker et a l . , 1985) and 159% i n the Djungarian hamster (Schlenker, 1985). In both species of ground s q u i r r e l , the increase i n V i n moderate hypoxia was achieved mainly through an increase i n f. Only i n severe hypoxia was there a contribution by increases i n V T (Figure 3). Walker et a l . (1985) reported that the Syrian hamster responded to 10% 0 2 by increasing f, with no change i n V T. In the same species, Holloway and Heath (1984) found that V T increased only when F I 0 2 was lowered to 13%. This response pattern was also observed i n the r a t , both awake and anaesthetized (Pappenheimer, 1977; Cragg and Drysdale, 1983). The hypoxic v e n t i l a t o r y responses of the Djungarian hamster, on the other hand, appears mediated by changes i n both f and V T (Schlenker, 1985). In S. l a t e r a l i s and S. columbianus f hypoxia caused decreases i n T T 0 T , T j (and so Tj/T^ rp ) and T E' (Table 2). This e f f e c t has also been reported f o r man (Dempsey and Forster, 1982), dogs (Ledlie et a l . , 1981), rats (Pappenheimer, 1977; Walker et a l . , 1985), and the Syrian hamster (Walker et a l . , 1985), but not f o r the Djungarian hamster (Schlenker, 1985). Despite the r e l a t i v e l y greater response of the Djungarian hamster to 57 10% 0 2, there was no a l t e r a t i o n of v e n t i l a t o r y timing, r e f l e c t i n g the greater contribution of V T to the v e n t i l a t o r y response i n t h i s species (Schlenker, 1985). In the studies of hamster v e n t i l a t o r y responses (Holloway and Heath, 1984; Schlenker, 1985; Walker et a l . , 1985), d i r e c t comparisons were made with e i t h e r the white r a t or the white mouse. The Djungarian hamster and white mouse appear to have a s i m i l a r hypoxic s e n s i t i v i t y , whereas the Syrian hamster, whose hypoxic response was cl o s e r to that of S. l a t e r a l i s and S. columbianus, has a higher hypoxic s e n s i t i v i t y than does the r a t (Holloway and Heath, 1984; Walker et a l . , 1985). Figure 10 i l l u s t r a t e s a comparison of the r e l a t i v e hypoxic responses of the two species of ground s q u i r r e l with those of the r a t . I t i s cl e a r that though both S. l a t e r a l i s and S. columbianus have a r e l a t i v e l y low r e s t i n g v e n t i l a t i o n compared to that of the r a t , the increase i n V/V 0 2 i s s i m i l a r i n a l l three species. Some of the v a r i a b i l i t y i n hypoxic responses can be ascribed to differences i n the exposure duration, which, i n various reports, ranges from 2 minutes to 2 hours. Moreover, Cragg and Drysdale (1983) point out that the f u l l expression of the hypoxic response of the anaesthetized r a t depends on the maintenance of isocapnia. During exposure to 15 or 10% 0 2, they found a decrease i n P a co2 ' w n ^ c ^ they could o f f s e t by addition of 6% C0 2 to the i n s p i r a t e . 58 Figure 10. Hey Plot showing hypoxic and hypercapnic v e n t i l a t o r y responses of Columbian and golden-mantled ground s q u i r r e l s awake at T A 22°C compared to those of the white r a t . Hypoxia: ground s q u i r r e l s 21,15,10,5, and 3% inspired 0 2; r a t 0 and 10% inspired 0 2. Hypercapnia: ground s q u i r r e l s 0,2,3,5, and 7% i n s p i r e d C0 2; r a t 0 and 5% inspired C0 2 Hypoxic responses are s i m i l a r i n a l l three species, but hypercapnic responses are comparatively reduced i n the ground s q u i r r e l s . Rat v e n t i l a t i o n data taken from Pappenheimer (1977). 59 V T ( ml / kg ) Walker e£ a l . (1985) also observed hypocapnia during hypoxic exposure i n the Syrian hamster and the r a t , though i t was l e s s severe i n the hamster. Since neither end-tidal nor a r t e r i a l blood gas tensions were measured i n the present study, the influence of hypocapnia on the hypoxic v e n t i l a t o r y responses of S. l a t e r a l i s and S. columbianus i s not known. The combination of 5% C0 2 and both 15 and 10% 0 2 enhanced the hypoxic response i n both S. l a t e r a l i s and S. columbianus awake at T A 5°C (Figure 9), so i t i s possible that hypocapnia occurred during exposure to these t e s t gases alone. I f so, the marked hyperventilation at F I 0 2 l e v e l s of 5% and 3% 0 2 would be expected to cause an even greater decrease i n P a c o 2 * I f t n * s occurred, then the v e n t i l a t o r y changes I have recorded may underestimate the true hypoxic responses. I t i s important to note, however, that t h i s would not detract from the conclusion that the two species have remarkably s i m i l a r v e n t i l a t o r y responses to hypoxia. The pattern of response to hyperoxia was also the same i n both species, and suggests that there i s a predominant hypoxic drive during air-breathing. I n s p i r a t i o n of 50% 0 2 decreased f and increased V T (Table 3). This would be expected i f hypoxia plays a major stimulatory r o l e at rest, since increasing PaQ2 w o u l d depress the hypoxic drive, reduce v e n t i l a t i o n , and allow P a c o 2 t o r ^ s e * The increase i n P aro2 would» i n turn, stimulate v e n t i l a t i o n mainly by increasing V T (Dejours, 1981). Such a speculation could be confirmed by measurements of end-tidal or a r t e r i a l blood gas tensions. In S. columbianus acutely exposed to T A 5°C, V was lower i n hyperoxia due to a decrease i n f. In conjunction with the higher r e s t i n g breathing frequency of t h i s group, t h i s suggests that cold exposure increases the hypoxic v e n t i l a t o r y drive. The i n a b i l i t y of cold-exposed animals to to l e r a t e 3% 0 2 supports t h i s , despite the fin d i n g that the v e n t i l a t o r y response to 5% 0 2 did not d i f f e r s i g n i f i c a n t l y between T A 22°C and T A 5°C (Figure 5). Conversely, during per i o d i c arousal, hyperoxia d i d not a l t e r V, though i t lead to a r i s e i n f and a f a l l i n V T. The reason f o r t h i s change i n breathing pattern i s not cle a r . In the absence of hypoxia, the V - P a C 0 2 response curve of most mammals i s l i n e a r (Dejours, 1981). Moderate decreases i n PaQ2 (>60 torr) s h i f t the C0 2 response curve upwards so that there i s a p o s i t i v e i n t e r a c t i v e e f f e c t that may be eit h e r additive (no synergism) or m u l t i p l i c a t i v e ( s y n e r g i s t i c ) . Further decreasing PaQ2 creates a negative i n t e r a c t i v e e f f e c t , due mainly to the depressant actions of cerebral ischaemia and hypoxia (Dempsey and Forster, 1982). S i m i l a r l y , increasing Paco2 tends to augment hypoxic s e n s i t i v i t y ( L a h i r i and Gelfand, 61 1981; Dempsey and Forster, 1982). In both S. l a t e r a l i s and S. columbianus, under a l l three conditions, the r e l a t i o n s h i p between V and F I C 0 2 was l i n e a r , and V rose through increases i n both f and V T (Figure 6). The o v e r a l l v e n t i l a t o r y response to hypercapnia was unaffected by cold exposure i n S. columbianus. but appears to be increased during pe r i o d i c arousal, due to a greater increase i n V T (Figure 8). The same response pattern to hypercapnia was also found i n the white r a t (Pappenheimer, 1977; L a i et a l . , 1978; Cragg and Drysdale, 1983), the Djungarian hamster and white mouse (Schlenker, 1985), the Syrian hamster (Chapin, 1954; Javaheri et a l . , 1980; Holloway and Heath, 1984; Walker et a l . , 1985), and the marmot (Leitner and Malan, 1973). The e f f e c t on f was comparatively greater i n these ground s q u i r r e l s than i n the hamsters (Schlenker, 1985; Walker et a l . , 1985), but i t was s t i l l not as great as the e f f e c t on V T (Figure 6). This i s borne out by the absence of changes i n v e n t i l a t o r y timing during hypercapnic exposure. Figure 10 shows the r e l a t i v e hypercapnic responses of the ground s q u i r r e l s compared to that of the r a t . The r e l a t i v e increase i n v e n t i l a t i o n i s c l e a r l y much lower i n both species of ground s q u i r r e l than i n the r a t . Blunted hypercapnic s e n s i t i v i t i e s have been reported f o r the Merriam's kangaroo r a t (Soholt et a_l., 1973), the mole rat ( A r i e l i and Ar, 1979), and the Syrian hamster (Chapin, 62 1954; Holloway and Heath, 1984; Walker et a l . , 1985), but not f o r the Djungarian hamster (Schlenker, 1985). Reduced hypercapnic s e n s i t i v i t y appears to be an adaptation to the f o s s o r i a l l i f e s t y l e , as i t i s found i n almost a l l burrowing mammals and birds (Boggs et a l . , 1984), and coincides with the high r e s t i n g P a c o 2 a n d * o w r e s t i n g v e n t i l a t i o n common to many burrowers ( A r i e l i and Ar, 1979; Boggs and Kilgore, 1984; Walker et a l . , 1985). The hypercapnia tolerance of burrowing animals may r e s u l t from an a l t e r a t i o n of chemoreceptor s e n s i t i v i t y and/or and increase i n buf f e r i n g capacity (Boggs et a l . , 1984). Further, i t appears to be ge n e t i c a l l y determined, since i n rats and mice chronic p e r i n a t a l C0 2 exposure does not a l t e r the adult hypercapnic v e n t i l a t o r y response (Birchard et a l . , 1984). The e f f e c t s of combined hypoxia and hypercapnia on v e n t i l a t i o n i n S. l a t e r a l i s and S. columbianus also reveal the hypercapnia tolerance of these ground s q u i r r e l s . No i n t e r a c t i o n was observed i n ei t h e r species u n t i l F I C 0 2 was increased to 5%; at t h i s C0 2 l e v e l there was a potentiation of the v e n t i l a t o r y responses at both 15% and 10% 0 2 (Figure 9). During peri o d i c arousal, t h i s e f f e c t i s enhanced, as i t occurs at 3% C0 2 as we l l . The Syrian hamster appears to show no i n t e r a c t i v e e f f e c t between hypoxia and hypercapnia, at l e a s t at 13% 0 2 and 7% C0 2 (Holloway and Heath, 1984). At 13% 0 2 and 5% C0 2, there i s a p o s i t i v e i n t e r a c t i o n i n the marmot, but i t i s l e s s than that of most non-hibernating mammals (Leitner and Malan, 1973). In contrast, the white r a t shows an additive e f f e c t at 15% 0 2 and 2% C0 2, but a negative i n t e r a c t i o n at 10% 0 2 and 8% C0 2 (Cragg and Drysdale, 1983). During acute co l d exposure, S. columbianus exhibited an i n t e r a c t i v e e f f e c t s i m i l a r to that of the r a t . Thus, i t appears that when MR i s (presumably) increased, the e f f e c t of high hypercapnia tolerance i s minimized, and the s q u i r r e l behaves more l i k e a non-fossorial animal. The r e l a t i v e l y greater hypercapnic response and the augmented hypoxia-hypercapnia i n t e r a c t i o n of the three S . columbianus tested during per i o d i c arousal from hibernation suggest that there has been some a l t e r a t i o n of v e n t i l a t o r y c o n t r o l . This e f f e c t i s presumably c e n t r a l , since not only was r e s t i n g breathing pattern altered, but also there was no change i n the hypoxic v e n t i l a t o r y response. However, confirmation of possible hibernation-induced changes i n v e n t i l a t o r y responses and control awaits further i n v e s t i g a t i o n i n a greater number of i n d i v i d u a l s . The importance of changes i n C0 2 s e n s i t i v i t y during hibernation w i l l be discussed i n more d e t a i l i n Chapter 4. Recording v e n t i l a t o r y responses to changes i n ins p i r e d gas composition can only give an estimate of the v e n t i l a t o r y s e n s i t i v i t y to hypoxia or hypercapnia. True v e n t i l a t o r y s e n s i t i v i t i e s cannot be quantified without measurements of end-tidal, or, preferably, a r t e r i a l blood 64 gas tensions. I t i s worthy of note, however, that examining the e f f e c t of in s p i r e d gases approaches the natural, p h y s i o l o g i c a l s i t u a t i o n more c l o s e l y i n burrowing animals than i n any others, since i n s p i r e d a i r i s the v e h i c l e by which these species are exposed to hypoxia and hypercapnia. With regard to hypoxia, high-altitude species experience a s i m i l a r s i t u a t i o n , but few other animals ever do. Thus the v e n t i l a t o r y responses of the ground s q u i r r e l s observed i n t h i s study would be predicted to mimic the behaviour of the animal i n i t s burrow. The v e n t i l a t o r y responses of S. l a t e r a l i s and S. columbianus and other f o s s o r i a l animals are well-suited to the problems of chronic exposure to an hypoxic/hypercapnic atmosphere. A reduced s e n s i t i v i t y to ambient hypercapnia, e s p e c i a l l y i n conjunction with an increased blood buffering capacity, would enable the burrower to withstand flu c t u a t i o n s i n i t s inspired a i r with minimum energy expenditure. This would be p a r t i c u l a r l y important i n a s i t u a t i o n i n which increased v e n t i l a t i o n would have no e f f e c t on lung C0 2 washout because F J C O 2 ^ s n e ^ d at a high l e v e l . Conversely, an i n t a c t hypoxic response, but a lowered threshold, may serve as a safeguard against hypoxic depression of v e n t i l a t i o n and metabolism. In addition, the upward s h i f t i n the minimum C0 2 l e v e l required to potentiate the hypoxic response would be b e n e f i c i a l to an animal that commonly experiences mild hypoxia and mild 65 hypercapnia concurrently. Burrowing animals are obliged to t o l e r a t e severe hypoxia and hypercapnia only i n extreme conditions. In t h i s l a t t e r s i t u a t i o n , the potentiation of the hypoxia-induced v e n t i l a t o r y response might serve to maintain P A 0 2 * n t n e face of an increase i n P a c o 2 a n d a decrease i n H b 0 2 binding a f f i n i t y . 66 CHAPTER THREE VENTILATORY RESPONSES IN HIBERNATING GROUND SQUIRRELS INTRODUCTION During entrance into hibernation, continuous breathing i s converted to intermittent breathing i n conjunction with the reduction i n metabolic rate and gas exchange requirements of the animal. In some species, including the hedgehog and dormouse, the intermittent breathing pattern i s characterized by long non-ventilatory, or apneic, periods interrupted by bursts of rapid v e n t i l a t i o n (Cheyne-Stokes r e s p i r a t i o n , CSR) (Kristoffersson and Soivio, 1964, 1966; Pajunen, 1970, 1974; Tahti, 1975; Tahti and Soivio, 1975). In others, such as the marmot, the apneas are separated by only one or two breaths (Endres and Taylor, 1930; Malan et a l . , 1973). The presence of these two d i s t i n c t l y d i f f e r e n t intermittent breathing patterns raises question as to whether there are species differences i n v e n t i l a t o r y control during hibernation. To date, studies of v e n t i l a t o r y control during hibernation have been l i m i t e d by the d i f f i c u l t i e s of measuring v e n t i l a t i o n i n undisturbed hibernating animals. The a v a i l a b l e evidence indicates that the hypoxia and hypercapnia tolerance c h a r a c t e r i s t i c of euthermic hibernators i s enhanced during hibernation. Almost a l l studies, however, report only changes i n breathing 67 frequency ( f ) . Hypercapnia i s known to have a strong e f f e c t on t i d a l volume (V T) i n non-hibernating mammals (Dejours, 1981), thus s e n s i t i v i t i e s based on f responses alone may underestimate the o v e r a l l v e n t i l a t o r y response. During hibernation, hamsters, ground s q u i r r e l s , marmots, and hedgehogs a l l increase f when the i n s p i r e d C0 2 l e v e l reaches 2-3% (Endres and Taylor, 1930; Lyman, 1951; Biorck et a l . , 1956; Tahti, 1975). Breathing becomes continuous at 6-7% F I C 0 2 i n the hedgehog (Tahti, 1975). In contrast, although f increases at an i n s p i r e d 0 2 l e v e l of 16% i n the hedgehog, breathing does not become continuous u n t i l i n s p i r e d 0 2 i s lowered to 3% (Tahti, 1975). These r e s u l t s suggest that changes i n blood "PC02 or pH play a greater r o l e i n the control of the intermittent breathing patterns displayed by hibernating animals (Tahti, 1975; Tahti and Soivio, 1975). As yet, however, there i s no evidence to explain the apparent species difference i n pattern of intermittent breathing. In part, t h i s i s due to the f a c t that most of the species that have been studied v i s a v i s v e n t i l a t o r y responses (notably the European hedgehog Erinaceus  europaeus) show CSR. There has been no previous comparison of the v e n t i l a t o r y responses of two species that display each of the two d i f f e r e n t intermittent breathing patterns. The purpose of t h i s part of the study was two-fold, as follows: 68 1. To quantify the o v e r a l l v e n t i l a t o r y responses of a hibernating animals to hypoxia and hypercapnia, including e f f e c t s on f, V T, and minute v e n t i l a t i o n (V), as well as on breathing pattern. 2. To compare the v e n t i l a t o r y responses of two species of ground s q u i r r e l that e x h i b i t each of the two breathing patterns to t e s t the hypothesis that the species difference i n intermittent breathing pattern may be explained on the basis of a difference i n s e n s i t i v i t y to hypoxia and hypercapnia. To t h i s end, the e f f e c t s of changes i n the l e v e l of i n s p i r e d 0 2 and C0 2, alone or i n combination, on v e n t i l a t i o n and breathing pattern were examined i n the golden-mantled ground s q u i r r e l (Sperroophilus l a t e r a l i s ) , which shows a Cheyne-Stokes breathing pattern, and the Columbian ground s q u i r r e l (S. columbianus), which takes only sing l e breaths. The r o l e s of changes i n 0 2 and C0 2 i n the control of intermittent breathing during hibernation w i l l be discussed. 69 MATERIALS AND METHODS Induction of hibernation Adult Columbian (Spermophilus columbianus) and golden-mantled (S. l a t e r a l i s ) ground s q u i r r e l s were induced to hibernate i n l a t e November of 1983 and 1984 by gradually reducing the ambient temperature (T A) and the l i g h t phase of the photoperiod i n the con t r o l l e d environment room i n which they were housed. Over about two weeks, T A was decreased from 2 0 + 1°C to 5 + 1°C and photoperiod changed from 12L:12D ( l i g h t s on 6AM) to 2L:22D ( l i g h t s on 10AM). During t h i s period the ground s q u i r r e l s had access to lab chow and water ad libitum, but were not handled or disturbed. Most animals had begun to hibernate before the end of the induction period, and a l l were hibernating within two weeks of exposure to cold and darkness. These environmental conditions were maintained throughout the winter. The ground s q u i r r e l s exhibited a l t e r n a t i n g bouts of hibernation and peri o d i c arousal. P e r i o d i c i t y of hibernation was not quantified, but appeared s i m i l a r to previous reports (bouts 1-2 weeks, arousal 24-48 hrs; Pengelley and Fisher, 1961; Twente and Twente, 1978). In l a t e A p r i l or early May of the following spring, T A and photoperiod were gradually returned to 20 + 1°C and 12L:12D respectively and the animals became active again. 70 Measurement of v e n t i l a t i o n The experimental arrangement used f o r recording v e n t i l a t i o n i n hibernating animals i s shown i n Figure 11. Breathing was measured using a small face mask made from the b a r r e l of a 50ml p l a s t i c syringe, molded on the i n s i d e with epoxy to minimize dead space. The mask was f i t t e d t i g h t l y over the animal's head with a rubber balloon. A small p l e x i g l a s s pneumotachograph was attached d i r e c t l y to the open end of the face mask. The t o t a l dead space of the face mask-pneumotachograph assembly did not exceed 0.3-0.4ml. The a i r f l o w across the pneumotachograph was registered by a d i f f e r e n t i a l pressure transducer (model DP103-18, Validyne, Northridge, C a l i f o r n i a ) , amplified (Gould transducer ampl i f i e r model 13-4615-50) and e l e c t r o n i c a l l y integrated (Gould integrator a m p l i f i e r model 13-4615-70 or Hewlett-Packard integrating preamplifier model 350-3700A) to give t i d a l volume (V T) d i r e c t l y . The V T s i g n a l was c a l i b r a t e d by i n j e c t i n g known volumes of a i r across the pneumotachograph v i a the face mask. Both a i r f l o w and V T signals were displayed continuously on a chart recorder (Gould 2400s or 2600 se r i e s recorder). In order to expose the hibernating animal to the t e s t gas mixtures, i t was placed into the animal chamber of the plethysmograph. In the laboratory, the chamber containing the hibernating s q u i r r e l was placed into a 500 cubic inch r e f r i g e r a t o r c o n t r o l l e d at 5 + 0.5°C. The volume of the Figure 11. Schematic diagram showing the experimental arrangement f o r recording v e n t i l a t i o n i n hibernating ground s q u i r r e l s . See text f o r description. 72 r e f r i g e r a t o r 0 N. to gas a n a l y z e r s \ < ^ W ) amplifier integrator / . — . A , ,chart ^recorder different ia l p r e s s u r e t r a n s d u c e r to g a s a n a l y z e r s f a c e - m a s k + p n e u m o t a c h o g r a p h animal chamber was 3.3 l i t r e s , t o t a l a i r flow was 1-2 liters/minute, and so turnover time of the chamber gas was 2-3 minutes. This was accounted f o r i n the determination of exposure duration. Test gases were created by mixing a i r with 100% C0 2, 0 2, or N 2 using flow meters. The gas composition of both inflow and outflow airstreams was analyzed to within +0.1% using Beckman OM11 0 2 and LB2 C0 2 gas analyzers, which were c a l i b r a t e d d a i l y with room a i r and premixed 5 and 10% C0 2 (Radiometer GMA2 p r e c i s i o n gas supply). The oxygen consumption (V Q 2) of hibernating animals was measured by placing the s q u i r r e l i n t o an a i r - t i g h t j a r ( i n the cold room) and taking two gas samples an hour apart. These gas samples were analyzed using the Beckman OM11 and LB2 gas analyzers, and V 0 2 was calculated by subtracting the f i n a l F I 0 2 from the i n i t i a l F I 0 2 and multiplying by t o t a l volume to give ml 0 2 consumed. The mean V Q 2 value f o r S. columbianus was 0.010 + 0.003 ml/g/hr (2 animals, 6 t r i a l s ) and f o r S. l a t e r a l i s i t was 0.021 + 0.011 ml/g/hr (5 animals, 13 t r i a l s ) ; these values were used to c a l c u l a t e V/V Q 2. Experimental protocol Hibernating animals were chosen f o r study only i f they had been i n hibernation f o r at l e a s t 24 hours. In preparation f o r the experiment, the animal chamber was taken int o the c o n t r o l l e d environment room and cooled. The 73 hibernating animal and some of i t s bedding were then placed into the chamber and body temperature (Tg) and heart rate (HR) leads connected. I f the ground s q u i r r e l showed no signs of arousal, the mask was placed over i t s snout and the balloon secured t i g h t l y over i t s head. Often t h i s procedure caused arousal. When i t di d not, however, the chamber containing the hibernating s q u i r r e l was moved to the r e f r i g e r a t o r i n the lab and l e f t f o r several hours to ensure that the animal stayed i n deep hibernation. Thereafter the s q u i r r e l s would hibernate wearing the mask f o r several days, demonstrating that the recording apparatus d i d not a f f e c t t h e i r a b i l i t y to hibernate. Once maintenance of deep hibernation was assured, a control breathing trace was recorded. The animal was then exposed to the t e s t gas mixtures (as described f o r awake animals; see Appendix I) i n random order and a l t e r n a t i n g sequence with a i r controls; the exposure duration was one hour, when a steady-state v e n t i l a t o r y response had occurred. Total recording time f o r the experimental t r i a l was approximately 36 hours. Chamber temperature ( T c ) , Tg, and HR (where possible) were monitored continuously throughout the experiment. T B and HR were used mainly as an i n d i c a t i o n of the depth of hibernation; Tg values ranged from 4.5-7 °C and experiments were not started unless HR was below 8-10 beats per minute (Wit and Twente, 1983). The animals were weighed only at the end of the t r i a l . Data analysis Breathing traces from both species were analyzed over about ten minutes of stable v e n t i l a t i o n at the end of the exposure period. The records were scored f o r o v e r a l l breathing frequency ( f ) , t i d a l volume (V T) and the duration of the non-ventilatory period (Tjjyp) • Minute v e n t i l a t i o n (V) was calculated as the product of f and V T. In addition, i n four S. l a t e r a l i s and f i v e S. columbianus. higher speed (1-5 mm/second) traces of approximately twenty breaths were analyzed f o r timing, including t o t a l breath duration ( T T 0 T ) , i n s p i r a t o r y time(Tj), and expiratory time (T E) (Figure 12). Breathing records from S. l a t e r a l i s were also analyzed for the following breathing pattern v a r i a b l e s : burst frequency (B/min), number of breaths per burst (b/B), burst duration (T Vp), and inter-burst non-ventilatory period (T E') (Figure 12) . Ven t i l a t o r y variables f o r a l l of the control periods were averaged to give experimental mean values f o r each s q u i r r e l ; these were then averaged to give a grand mean f o r each species. Data presented as percent change (e.g. %A V/V 0 2) were calculated as the percent change from control f o r each t e s t gas f o r each animal, and then averaged to give mean percent change f o r each species. S t a t i s t i c a l analysis was as described i n Chapter Two. 75 Figure 12. Representative breathing records i l l u s t r a t i n g timing and breathing pattern v a r i a b l e s . The t y p i c a l breathing pattern of the hibernating Columbian ground s q u i r r e l i s shown on the l e f t , and that of the golden-mantled ground s q u i r r e l on the r i g h t . The small d e f l e c t i o n s on the a i r flow traces shown here are synchronous with heart beats. 76 T T O T = total breath duration T| = inspiratory time T E = expiratory time T V P = ventilatory period T E ' = intra-burst non-ventilatory period T N V P = inter-burst non-ventilatory period RESULTS Resting v e n t i l a t i o n Representative control breathing traces f o r both species of ground s q u i r r e l are shown i n Figure 13. The corresponding mean v e n t i l a t o r y variables f o r 7 animals of each species are l i s t e d i n Table 4. An intermittent breathing pattern was observed i n a l l hibernating animals, although the two species exhibited d i s t i n c t l y d i f f e r e n t patterns. The Columbian ground s q u i r r e l took infrequent s i n g l e breaths followed by 1-2 minute non-ventilatory periods (mean value 70.8 + 4.3 seconds, maximum value 190 seconds). In contrast, the golden-mantled ground s q u i r r e l took bursts of several breaths (mean value 6.86 + 0.58) separated by longer non-ventilatory periods, ranging from le s s than one minute to greater than t h i r t y minutes (mean value 115.8 +25.3 seconds, maximum value 32 minutes). Both species displayed considerable v a r i a b i l i t y i n r e s t i n g breathing pattern; i n d i v i d u a l animals tended to show les s v a r i a b i l i t y over the course of a recording session. Overall f ranged from 0.50 to 2.21 i n S. columbianus and from 1.29 to 3.54 i n S. l a t e r a l i s . The v a r i a b i l i t y of breathing pattern was more obvious i n S. l a t e r a l i s ; B/min ranged from 0.21 to 0.90; b/B from 3 or 4 to greater than 30. This v a r i a b i l i t y did not appear to be related to T A or T B, and as 0 2 Figure 13. Representative records of the r e s t i n g breathing patterns i n hibernating ground s q u i r r e l s at T A of 5°C. Note that t i d a l volume i s approximately twice as great i n the Columbian ground s q u i r r e l as i n the golden-mantled ground s q u i r r e l . 78 Golden-mant led ground squirre l Air Flow Volume (ml) i n out •10 r -1.0 -Columbian ground squ i r re l 1min in Air Flow Volume (ml) out •2.0r -2.0 44 4-4-4 TABLE 4. Ventilatory and breathing pattern variables in hibernating golden-mantled (S. lateralis) and Columbian (S.columbianus) ground squirrels during air exposure. A l l values are mean standard error. See text for explanation of symbols. VARIABLE S. latera l i s S. columbianus n 7 7 mass (gm) 195.8 • 26.1 304.1 • 23.8 Tft(°C) 5.0 • 0.5 5.0 i 0.5 V <ml/min) 2.51 * 0.43 1.79 •. 0.11 VT <»1) 0.98 0.10 1.76 1 0.11 f (b/min) 2.59 0.30 1.14 i 0.09 TTOT (sec) 4.76 • 0.12 6.25 • 0.08 Ti (sec) 1.76 • 0.05 2.50 1 0.08 T£ (sec) 3.01 • 0.08 3.75 • 0.05 Tg' (sec) 6.2 • 0.53 -T(JVP (sec) 115.8 25.3 70.8 1 4.3 Ty/P (sec) 59.0 • 9.8 -B/min 0.47 • 0.09 -b/B 6.86 0.58 -79 consumption (V Q 2) was not measured simultaneously with v e n t i l a t i o n , any causal r e l a t i o n s h i p between the v a r i a b i l i t i e s of breathing pattern and V 0 2 cannot be determined. Overall f was approximately twice as high i n S. l a t e r a l i s as i n S. columbianus but V T was somewhat lower, so that V was only 1.4 times higher i n the former smaller species (see Table 1). S. columbianus took much slower breaths than d i d S. l a t e r a l i s ; however, the r a t i o of mean Tj to T T 0 T (duty cycle) was s i m i l a r f o r both species, being, on average, 0.40 i n the former and 0.37 i n the l a t t e r . Responses to hypoxia Inhalation of hypoxic gas mixtures had l i t t l e e f f e c t on v e n t i l a t i o n and breathing pattern i n both species of hibernating ground s q u i r r e l (Figure 14). The v e n t i l a t o r y responses of the two species to decreased i n s p i r e d 0 2 are plo t t e d i n Figure 15. Both S. columbianus and S. l a t e r a l i s showed very l i t t l e change i n f, V T, or V even at very low 0 2 l e v e l s . Only S. l a t e r a l i s was found to show any v e n t i l a t o r y increase at 5 and 3% inspired 0 2; V rose from the control value of 2.51 to 4.65 ml/minute at 3% 0 2, due to a s i g n i f i c a n t increase i n o v e r a l l f from 2.59 to 4.9 breaths/minute. In contrast, v e n t i l a t i o n decreased 80 s l i g h t l y i n S. columbianus. from 1.79 ml/minute to 1.33 ml/minute, corresponding to a change i n f from 1.14 breaths/minute to 0.92 breaths/minute. Although V T was higher i n S. columbianus at a l l 0 2 l e v e l s , neither species experienced a s i g n i f i c a n t change i n V T during hypoxic exposure. The tendency towards v e n t i l a t o r y depression seen i n S. columbianus i n severe hypoxia was also observed i n S. l a t e r a l i s at 3% 0 2. Four S. l a t e r a l i s and two S. columbianus were tested at an F I 0 2 of 1% with v a r i a b l e r e s u l t s . Two S. l a t e r a l i s showed an increase i n f and V and subsequent arousal from hibernation. The remaining four animals showed a decrease i n f and V, and subsequent metabolic depression. In two cases, the metabolic depression was so profound that i t probably would have caused death had the s q u i r r e l s not been removed from the chamber and resuscitated. The e f f e c t s of hypoxia on breathing pattern variables are shown i n Figure 16. S. l a t e r a l i s continued to show CSR at a l l l e v e l s of inspir e d 0 2, though mean burst frequency was increased s l i g h t l y above the control l e v e l i n severe hypoxia (panel a). In t h i s species, an increase i n b/B and concurrent increase i n T v p contributed to the r i s e i n o v e r a l l f at 5 and 3% 0 2 (panels b and c ) , although the greatest change was a decrease i n the T^yp (panel d ). In S_. columbianus, T^yp was s l i g h t l y longer i n severe hypoxia, leading to the decrease seen i n f (panel d ) . 81 Figure 14. E f f e c t s of hypoxia and hypercapnia on breathing pattern during hibernation. Intermittent breathing p e r s i s t e d during exposure to 5% inspir e d 0 2, but was converted to continuous breathing during exposure to 5% C0 2 i n both species. In the golden-mantled ground s q u i r r e l , the p e r i o d i c i t y of v e n t i l a t i o n often remained even when breathing was continuous. 82 Golden - mantled ground squirrel AIR E ~ 5 % 02 p i E 1 1.0 [ ^ A A J l A A ^ A JJLAAJU J L 2 5 % CO2 i . 0 [ X l V U J L A i i J J U J l A ^ L J L Columbian ground squirrel AIR 5 % o 2 [ A _ _ J A _ J — h -5 % co 2 [JUJU'JUJULiJJLLU 1 J 30 sec Figure 15. E f f e c t s of decreasing F j 0 2 on minute v e n t i l a t i o n (V), t i d a l volume (V T), and breathing frequency (f) i n hibernating Columbian ( • ) and golden-mantled ( A ) ground s q u i r r e l s . A l l values are mean + standard error f o r 5-7 animals. 83 ( m l / m i n ) Figure 16. E f f e c t of decreasing F I 0 2 o n breathing pattern v a r i a b l e s i n hibernating Columbian ( • ) and golden-mantled ( A ) ground s q u i r r e l s . B/min = burst frequency; b/B = breaths per burst; T v p = v e n t i l a t o r y period; Tjjyp = non-ventilatory period. A l l values are mean + standard error f o r 5-7 animals. 84 T Vp (sec) B/min Hypoxia had no s i g n i f i c a n t e f f e c t on the timing of i n d i v i d u a l breaths i n e i t h e r species; nor d i d i t a l t e r the length of T E' within the bursts of S. l a t e r a l i s (Table 6 ) . Responses to hypercapnia Both species of ground s q u i r r e l showed strong v e n t i l a t o r y responses to increased i n s p i r e d C0 2. The response threshold f e l l between 2 and 3% i n s p i r e d C0 2, a f t e r which the response curve rose steeply (Figure 17). In the S. l a t e r a l i s V increased from the control value of 2.51 ml/minute to 13 ml/minute at 5% C0 2; i n S. columbianus. i t increased from 1.79 to 9.0 ml/minute. At 7% C0 2, V had increased further to 11.2 ml/minute i n S. columbianus, but had f a l l e n s l i g h t l y to 11.9 ml/minute i n S. l a t e r a l i s . There was no s i g n i f i c a n t difference i n V between the two species at any l e v e l of inspir e d C0 2. In both species, the increase i n V was due to increases of both f and V T (Figure 17). At 7% i n s p i r e d C0 2, V T increased from the r e s t i n g value of 1.76 ml to 2.5 ml i n S. columbianus and from 0.99 ml to 1.45 ml i n S. l a t e r a l i s . O v e r all f increased from 1.14 breaths/minute to 4.4 breaths/minute i n the former species; i n the l a t t e r , i t rose from 2.59 breaths/minute to 8.7 breaths/minute. Regardless of r e s t i n g intermittent breathing pattern, the increase i n f during hypercapnic exposure was due to a 85 Figure 17. E f f e c t s of increasing F I C 0 2 on minute v e n t i l a t i o n (V), t i d a l volume ( V T ) , and breathing frequency (f) i n hibernating Columbian ( • ) and golden-mantled ( A ) ground s q u i r r e l s . A l l values mean + standard error f o r 5-7 animals. 86 V ( m l / m i n ) Figure 18. E f f e c t of increasing F I C 0 2 o n breathing pattern v a r i a b l e s i n hibernating Columbian (• ) and golden-mantled ( A ) ground s q u i r r e l s . B/min = burst frequency; b/B = breaths per burst; T v p «= v e n t i l a t o r y period; Tj^yp = non-ventilatory period. The hatched l i n e and "cont." indicate the point at which breathing became continuous i n the golden-mantled ground s q u i r r e l . A l l values are mean + standard error f o r 5-7 animals. 87 T y p (sec) B/min OD " T N V P (sec) b / B decrease i n Tjjyp (Figure 18, panel d) from 70.8 seconds to 8.80 seconds i n S. columbianus and 115.8 seconds to 4.67 seconds i n S. l a t e r a l i s at 7% C0 2. Below 5% C0 2, most S. l a t e r a l i s continued to breathe i n bursts, though as Tjjyp f e l l , burst frequency increased (Figure 18, panel a). There was a s l i g h t increase i n b/B and a decrease i n T Vp. At 5 and 7% C0 2, burst breathing was converted to continuous breathing i n a l l i n d i v i d u a l s (Figures 14 and 18). The p e r i o d i c i t y of V T often remained even a f t e r breathing had become continuous. The CSR pattern of v e n t i l a t i o n i n v a r i a b l y reappeared upon return of the animal to a i r . As with hypoxia, hypercapnia had no s i g n i f i c a n t e f f e c t on the timing of i n d i v i d u a l breaths i n e i t h e r species, though i n S. columbianus. T T 0 T tended to decrease with increased F I C 0 2 , due to a shortening of T j (Table 6). Responses to hyperoxia, hyperoxic hypercapnia, and hypoxic hypercapnia Table 5 summarizes the e f f e c t s of hyperoxic, hyperoxic/hypercapnic, and hypoxic/hypercapnic gas mixtures on v e n t i l a t i o n i n both species of ground s q u i r r e l . The values of V f o r a l l l e v e l s of inspir e d 0 2 and C0 2 are shown i n Figure 19. Increasing the inspir e d 0 2 f r a c t i o n to 50% had no s i g n i f i c a n t e f f e c t on breathing pattern or o v e r a l l l e v e l of 88 v e n t i l a t i o n i n e i t h e r species (Tables 5 and 7). While there was no change i n the timing of i n d i v i d u a l breaths i n S. columbianus, S. l a t e r a l i s showed a small, but i n s i g n i f i c a n t , decrease i n both T T O f r and T j during exposure to hyperoxia (Table 6). In most cases, changing the l e v e l of i n s p i r e d 0 2 also had no e f f e c t on the v e n t i l a t o r y response to hypercapnia i n hibernating animals. The increase i n V from 0 to 3 and 5% C0 2 was the same i n S. l a t e r a l i s regardless of the concomitant l e v e l of i n s p i r e d 0 2. This was also true f o r S. columbianus at 3% C0 2 and at 5% C0 2 down to 15% 0 2. At 10 and 5% i n s p i r e d 0 2 there was some decrease i n the response to 5% C0 2 i n t h i s species. The values obtained at 5% 0 2 and 3 and 5% C0 2 are based on only two S. l a t e r a l i s and three S. columbianus. so i t i s d i f f i c u l t to determine the v a l i d i t y of t h i s trend i n S. columbianus. Similar to the v e n t i l a t o r y responses to hypercapnia alone, the increase i n o v e r a l l f was due mainly to a decrease i n Tjjyp i n both species (Table 7) . In S. l a t e r a l i s v e n t i l a t i o n became continuous at 3 to 5% i n s p i r e d CC*2; only one i n d i v i d u a l persisted to burst-breathe at 15% 0 2 and 5% C0 2. Some v a r i a b i l i t y of the changes of breathing pattern was observed i n t h i s species. At 3% i n s p i r e d C0 2 and a l l l e v e l s of 0 2 l i s t e d i n Table 6, about one-half of the s q u i r r e l s continued to burst-breathe, 89 though Tjjyp was decreased, and so b/min increased, compared to co n t r o l . Again, V T v a r i a b i l i t y was often observed even when breathing became continuous, and CSR reappeared upon return to a i r . The e f f e c t s of hyperoxic and hypoxic hypercapnia on v e n t i l a t o r y timing are shown i n Table 6. In both S. columbianus and S. l a t e r a l i s , there was no s i g n i f i c a n t change i n T T 0 T , T j , or T E, although at 5% C0 2 and 50, 15, and 10% 0 2 both T T 0 T and T j tended to decrease (Table 6). 90 TABLE 5. E f f e c t s o f h y p e r o x i c , h y p e r o x i c / h y p e r c a p n i c , and h y p o x i c / h y p e r c a p n i c g a s m i x t u r e s on b r e a t h i n g f r e q u e n c y ( f , b r e a t h s / m i n u t e ) , t i d a l volume (VT , m l ) , and m i n u t e v e n t i l a t i o n ( V , m l / m i n u t e ) i n h i b e r n a t i n g g o l d e n - m a n t l e d <S. l a t e r a l i s ) and C o l u m b i a n ( S . c o l u m b i a n u s ) g r o u n d s q u i r r e l s . A l l v a l u e s a r e mean s t a n d a r d e r r o r . GAS VARIABLE n S . l a t e r a l i s n S . c o l u m b i a n u s MIXTURE 50*02 0XC02 50*02 3XC02 15X02 3XC02 10X02 3XC02 5X02 3XC02 50X02 5XC02 15X02 5XC02 10X02 5XC02 5X02 5XC02 f VT V f VT V f VT V f VT v f VT v f VT v f v T V f VT v f YT v 2.89 0.80 6 1.17 0.33 0.97 + 0.13 1.47 • 0.24 2.87 0.86 1.42 • 0.27 5.89 • 1.08 4 2.91 • 0.84 1.11 • 0.11 1.61 • 0.31 6.19 • 0.87 4.33 • 0.74 6.39 0.99 5 1.88 0.33 1.12 • 0.08 2.05 0.26 7.00 + 1.13 3.55 0.33 6.81 • 0.96 5 1.92 0.33 1.08 0.21 1.95 • 0.23 6.67 • 2.63 3.54 •4- 0.48 4.06 1.00 3 2.12 • 0.24 1.30 0.04 1.57 • 0.40 5.17 • 1.14 3.18 • 0.58 8.03 1.91 5 3.99 • 0.70 1.40 • 0.14 2.29 • 0.34 L1.09 • 2.08 6.48 • 1.32 8.91 • 1.00 5 3.65 + 0.52 1.37 * 0.24 2.60 • 0.25 L0.98 1.07 9.32 + 1.38 9.50 1.07 6 3.14 0.42 1.55 0.25 2.42 • 0.18 L3.25 • 0.64 7.45 • 0.93 8.60 • 0.99 3 3.30 0.46 1.48 • 0.07 1.92 • 0.42 L2.87 2.07 5.95 • 0.67 91 Figure 19. Bar p l o t showing the e f f e c t s of changes i n FjQ2 a n d FIC02' a l ° n e o r * n combination, on minute v e n t i l a t i o n (V) i n hibernating ground s q u i r r e l s , n = 6 for a l l cases except 5% 0 2 and 3 or 5% C0 2, where n = 3 f o r the Columbian ground s q u i r r e l and n = 2 f o r the golden-mantled ground s q u i r r e l . The v e r t i c a l l i n e on each bar represents one standard error of the mean. 92 Columbian ground squirrel El Go lden-mant led ground squirrel • 1(H 5- 0 3 Z . © CN o o LL ''lit! I v * | i l : l | '»"/:: It It 50 21 15 F i o 2 <0/*> 10 TABLE 6. Effect of alteration of inspired gas composition on the ventilatory timing variables total breath duration <TT0T» seconds), inspiratory time (Tx, seconds), expiratory time (Tg, seconds), and end-expiratory pause (Tg', seconds) in hibernating golden-mantled (S. lateralis) and Columbian <S. columbianus) ground squirrels. A l l values are mean • standard error. GAS VARIABLE n S. lat e r a l i s n S. columbianus control TTOT 6 4.76 0 . 1 2 5 6.22 0 . 1 3 TI 1.76 0.05 2.42 0.22 TE 3.01 * 0.08 3.80 • 0.14 TE' 6.20 0 . 5 3 1 5 X 0 2 TTOT 4 4.81 0.48 5 6.00 • 0.38 TI 1.80 0 . 1 8 2.26 + 0 . 2 3 TE 3.04 0.32 3.73 • 0.26 TE' 6.46 • 2.7 1 0 X 0 2 TTOT 3 4.42 • 0 . 7 3 4 6.53 • 0.64 TI 1.39 0 . 1 0 2 . 7 3 0.57 TE 3 . 0 3 • 0.63 3.80 + 0 . 1 1 TE' 4 . 8 3 1.91 5 X 0 2 TTOT 4 4. 7 3 0.56 4 6.76 0.27 TI 1.82 • 0.23 3.06 * 0.38 TE 2 . 9 1 • 0.36 3.96 0.12 TE' 7 . 3 5 2.7 3 X 0 2 TTOT 5 4.31 0.42 3 6.53 0.32 TI 1.61 • 0 . 1 5 2.63 0.45 TE 2 . 7 0 • 0.24 3.90 0.49 2 X C 0 2 TTOT 4 4.50 • 0.46 4 6.20 0.25 TI 1.64 • 0 . 1 5 2.37 0.31 TE 2.87 • 0.32 3.81 * 0.14 3 X C 0 2 TTOT 5 4 . 1 5 + 0.27 4 5.83 * 0.23 TI 1.58 • 0 . 1 3 2.26 0.26 TE 2.57 + 0 . 1 5 3 . 5 5 0.10 5 X C 0 2 TTOT 3 4.27 • 0.92 4 5.68 0 . 1 9 TI 1.66 • 0.36 1.90 • 0 . 1 1 TE 2.64 • 0.58 3.78 • 0 . 1 9 7 X C 0 2 TTOT 5 5.08 0.64 4 5.63 0.34 TI 1.78 0.26 1.93 • 0.22 TE 3.28 • 0.47 3.63 0.34 9 3 T a b l e 6, c o n t i n u e d . . . 50X02 TTOT 5 4.36 • 0 .48 4 6.26 0.24 TI 1.43 • 0.17 2.41 • 0.30 TE 2.94 • 0.39 3.83 + 0.20 TE' 5.53 + 0.83 50X02 TTOT 5 4.36 • 0.36 4 5.53 • 0 .43 3XC02 TI 1.52 • 0.09 2.05 0.34 TE 2.85 + 0.24 3.51 * 0.23 TE' 4.36 • 1.03 15X02 TTOT 4 4.25 0.50 3 5.77 + 0.45 3XC02 TI 1.53 0.20 2.27 + 0.36 TE 2.75 • 0.33 3.50 0.25 10X02 TTOT 4 4.76 • 0.82 3 6.12 • 0.22 3XC02 TI 1.86 • 0 .40 2.28 • 0.42 TE 2.90 • 0.44 3.83 • 0.21 5X02 TTOT 2 5.12 0.09 3 6.75 0.23 3XC02 TI 1.82 • 0.18 2.77 0.15 TE 3.22 0.02 4.00 • 0.36 50X02 TTOT 5 4.86 0.75 4 5.74 • 0.37 5XC02 TI 1.75 0.36 1.90 + 0.25 TE 3.11 • 0.39 3.87 + 0.14 15X02 TTOT 4 4.46 0.66 3 5.74 0.42 5XC02 TI 1.69 0.37 1.98 • 0.54 TE 2.77 * 0.30 3.77 0.30 10X02 TTOT 4 4.20 • 0.64 4 5.62 0.33 5XC02 TI 1.57 0.24 2.62 • 0.40 TE 2.62 0.40 3.45 • 0.24 5X02 TTOT 2 4.52 • 0.37 3 6.31 • 0.16 5XC02 TI 1.61 0.15 2.37 • 0.09 TE • 2.91 0.22 3.93 0.20 9 4 TABLE 7. E f f e c t s of hyperoxic, hyperoxic/hypercapnic, and hypoxic/hypercapnic gas mixtures on breathing pattern variables i n hibernating golden-mantled (S. l a t e r a l i s ) and Columbian (S. columbianus) ground s q u i r r e l s . A l l values are mean standard e r r o r . See text f o r explanation of symbols, n = t o t a l number of animals; m = number of animals showing CSR (bursting); a denotes values f o r CSR; c denotes values f o r continuous breathing. S. l a t e r a l i s S. columbianus GAS n/m B/min b/B Tvp TfiVP n TjuVP MIXTURE (sec) (sec) (sec) 50X02 7/7 0.36 7.8 65.9 135.8 5 81.5 0XC02 1 0.07 • 2.0 • 8.1 • 41.0 • 27.3 50X02 5/3 0.68 7.1 54.5 a 47.2 4 19.0 3XC02 1 0.35 • 0.66 • 18.3 • 17.8 4.6 c 4 .8 1 2.2 15X02 7/4 1.22 4.6 36.4 a 25.1 5 30.6 3X02 1 0.28 • 0.5 • 14.1 • 7.1 • 3.8 10X02 7/3 0.67 8.1 47.3 a 39.1 5 30.0 3XC02 i. 0.12 • 3.4 • 12.0 • 8.9 • 7.2 c 3.4 1 1.0 5X02 2/0 - - - 5.6 3 23.3 3XC02 1 0.8 4.5 50X02 5/0 - 3.5 5 11.8 5XC02 • 1.0 i 3.7 15X02 7/1 0.42 10.8 119.0 a 35.0 5 12.0 5XC02 c 3.4 • 3.1 1 1.4 10X02 7/0 - 2.4 6 14.3 5XC02 1 0.9 i 2.4 5X02 2/0 - 3.3 3 11.7 5XC02 1 0.5 •_ 2.6 95 DISCUSSION The major fi n d i n g of t h i s part of the study i s that hibernating Columbian and golden-mantled ground s q u i r r e l s have nearly i d e n t i c a l v e n t i l a t o r y responses to both hypoxia and hypercapnia. This i s true regardless of the r e s t i n g intermittent breathing pattern and thus the r e s u l t s do not support the hypothesis that the difference i n pattern of intermittent breathing may be explained on the basis of a d i f f e r e n c e i n s e n s i t i v i t y to respiratory gases. Furthermore, the v e n t i l a t o r y responses to hypoxia and hypercapnia of hibernating S. columbianus and S. l a t e r a l i s substantiate the claim of Tahti and coworkers (Tahti, 1975; Tahti and Soivio, 1975; Tahti et a l . , 1981) that intermittent breathing during hibernation i s c o n t r o l l e d mainly by changes i n a r t e r i a l Pco2 o r P H' r a t h e r than i n p 0 2 * The breathing patterns and v e n t i l a t o r y v a r i a b l e s recorded here compare well with previously reported values f o r hibernating animals (Table 8 ). To my knowledge, t h i s i s the f i r s t report of breathing pattern i n hibernating Columbian ground s q u i r r e l s ; a l l previous investigators also observed CSR i n hibernating golden-mantled ground s q u i r r e l s (Hammel et a l . , 1968; Steffen and Riedesel, 1 9 8 2 ) . Since CSR i s known to disappear i f the animal i s disturbed (Pembrey and P i t t s , 1899; K r i s t o f f e r s s o n and Soivio, 1964; Pajunen, 1970; Tahti, 1975), t h i s agreement indicates that the use of the face-mask and pneumotachograph had no detrimental e f f e c t on breathing pattern. The breathing pattern variables recorded f o r S. l a t e r a l i s d i f f e r somewhat from those of Hammel et a l . (1968) and Steffen and Reidesel (1982) (see Table 8). S p e c i f i c a l l y , Tjjyp tended to be shorter, averaging 2-3 minutes, compared to 8 minutes i n the l a t t e r study. This may r e f l e c t differences i n T A, since Hammel et a l . (1968) observed that breathing pattern i n hibernating S. l a t e r a l i s was profoundly affected by changes i n T A and i n hypothalamic temperature ( T H y P 0 ) . At higher T A's (6-8°C) and Tjjypn's (5.5-15°C), the pattern consisted of bursts of 10-30 breaths separated by 5-10 minute non-ventilatory periods. As T A f e l l below 4°C, corresponding to T H y P 0 below 5°C, both b/B and Tjjyp decreased, so that at a T H y p 0 o f 3.5°C breathing was almost continuous (Hammel et a l . , 1968). Endres and Taylor (1930) observed a s i m i l a r e f f e c t of T B on breathing pattern i n the marmot, as di d K r i s t o f f e r s s o n and Soivio (1964) i n the hedgehog and Pajunen (1970) i n the dormouse. Consequently, i t i s d i f f i c u l t to compare r e s u l t s obtained at d i f f e r e n t T A's. Since Steffen and Riedesel (1982) kept T A at 8°C, t h e i r longer Tj^yp values are not unexpected. In the 13-lined ground s q u i r r e l at T A 5°C, Landau and Dawe (1958) found that Tj^yp could vary from a few seconds to about 5 minutes (Table 8). These values are s i m i l a r to those measured here i n S. l a t e r a l i s and S. columbianus at the same T A. As well, several studies report marked v a r i a b i l i t y i n breathing frequency even at constant T A. Lyman (1951) observed i n the golden hamster that f varied as much as 25% from the mean during a sing l e recording session. S i m i l a r l y , he reported a range of 0.3 to 16 breaths/minute i n the 13-lined ground s q u i r r e l (Table 8). In the woodchuck and the y e l l o w - b e l l i e d marmot, f may vary between 0.3 and 7 breaths/minute (Goodrich, 1973); i n the European marmot Malan et a l . (1973) recorded Tjjyp values ranging from 1 to 6 minutes (Table 8). Moreover, Malan et a l . (1973) measured V 0 2 and v e n t i l a t i o n simultaneously i n hibernating marmots using whole body plethysmography and found no c o r r e l a t i o n of f with V Q 2 . Higher f values were not coincident with high V 0 2 values, nor with higher T B values, suggesting that the animals showing higher breathing frequencies were s t i l l i n deep hibernation. Steffen and Riedesel (1982) obtained V T values f o r S. l a t e r a l i s that were higher than those measured here (11.6 ml/kg, or 2.3 ml i n a 200gm animal, compared to 0.98 ml). The reason f o r t h i s difference i s not c l e a r , but i t may r e f l e c t the higher T A used by these authors. Perhaps at higher T A, S. l a t e r a l i s breathes more slowly but takes deeper breaths, while at lower T A i t takes more frequent, 98 TABLE 8. Ventilatory variables in various species of hibernating manuals. SPECIES TA T B PATTERN f VT V THVP b/B SOURCE oc oc br ml ml min min min hamster 4-7 0.46 Lyman, 1951 Meso-cricetus auratus 4 CSR 8 6-8 Kristoffersson and Soivio, 1964 it 10 CSR 1 several " dormouse 11-16 CSR 3.0 3-4 4-14 Pembrey and Hyoxus Pitts,1899 avellanarius ground squirrels S. t r i - 4-7 0.3-16 Lyman, 1951 decem-lineatus 5 single 1-3 few sec- 1-2 Landau and 3-5 min Dawe, 1958 S. >4 CSR 5-10 5-10 Hammel et al_., la t e r a l i s 1968 8 CSR 2.1 11.6 24.4 8-9 Steffen and ml mi/ka. Riedesel, 1982 kg min hedgehog Erinaceus 2-7 CSR 13 Biorck et al,., europaeus 1956 4.2 CSR 67-78 Tahti, 1975 4.2 CSR 50-70 Tahti and Soivio, 1975 marmots 10-12 single long Pembrey and Pitts, 1899 4.8 single 0.33 5.6 1.85 3 Endres and Taylor, 1930 8.1 single 1.0 13.8 13.8 1 • t M.monax 3.2- 2.4 Goodrich, 1973 M.flavi- 8.3 (0.3-7.0) ventris M. 5 single 0.93 32.5 30.3 1.6 Malan et a l . , marmota (11.9-48.9) 1973 99 shallower breaths. In addition, the o v e r a l l V was higher i n t h e i r study (24.4 ml/kg/min, or 4.88 ml/min i n a 200gm animal, compared to 2.51 ml/min), as was HR, which may in d i c a t e that the animals of Steffen and Riedesel (1982) were not hibernating as deeply as those studied here. Changes i n the inspir e d 0 2 l e v e l , whether hypoxic or hyperoxic, had very l i t t l e e f f e c t on v e n t i l a t i o n and breathing pattern i n both S. l a t e r a l i s and S. columbianus. With the exception of the v e n t i l a t o r y response to 3% 0 2, t h i s was true regardless of the r e s t i n g intermittent breathing pattern. The importance of the difference i n response to 3% 0 2 w i l l be addressed l a t e r i n t h i s discussion. Hibernating golden-mantled and Columbian ground s q u i r r e l s appear to have a lower hypoxic s e n s i t i v i t y than does the hedgehog, i n which Tj^p was found to shorten at an F I 0 2 of 16% (Tahti, 1975). Breathing became continuous at 3% 0 2 i n the hedgehog, but d i d not i n e i t h e r S. l a t e r a l i s or S. columbianus (Figure 16). The remarkably low hypoxic v e n t i l a t o r y responses i n these s q u i r r e l s during hibernation may be explained i n part by the f a c t that metabolic rate (MR) i s decreased to 1/3 0 to 1/50 of the euthermic l e v e l (Wang, 1978; Malan, 1982), and that the hemoglobin-oxygen (Hb02) d i s s o c i a t i o n curve i s s h i f t e d dramatically to the l e f t when T B f a l l s to 5°C. In the 13-lined ground s q u i r r e l , P 5 0 f a l l s from 36 i d o t o r r at 38°C to about 6.5 t o r r at 6°C (Musacchia and Volkert, 1971); i n the hedgehog i t i s 34 t o r r at 38°C and 8.9 t o r r at 5°C (Clausen and Ersland, 1968). This decrease i n P 5 0 corresponds to an increase i n 0 2 binding a f f i n i t y , and as such, to a saturation of a r t e r i a l blood at very low ambient 0 2 l e v e l s . Figure 20 shows the re l a t i o n s h i p between the hypoxic v e n t i l a t o r y responses of the two ground s q u i r r e l species, both awake and hibernating, and the corresponding Hb0 2 d i s s o c i a t i o n curves f o r blood at 37°C and 5°C. At 37°C, the hypoxic response threshold of about 10% 0 2 corresponds to the shoulder of the d i s s o c i a t i o n curve, so that as P 0 2 begins to f a l l , v e n t i l a t i o n increases (and presumably P a 0 2 i s maintained). Note that the steepest portion of the v e n t i l a t o r y response curve occurs c o i n c i d e n t a l l y with the steepest portion of the d i s s o c i a t i o n curve, suggesting that the increase i n V and the decrease i n saturation are matched. When T B f a l l s to 5°C during hibernation, the Hb0 2 curve i s s h i f t e d so f a r to the l e f t and becomes so steep that, i f the above ra t i o n a l e i s followed, a v e n t i l a t o r y response would not be expected u n t i l P 0 2 f a l l s to 10-15 t o r r (Figure 20). A f t e r t h i s point, P 0 2 f a l l s so r a p i d l y that any increase i n v e n t i l a t i o n may not compensate, and metabolic depression ensues. This was observed i n both species at 1% inspired 0 2, which would 101 Figure 20. Relationship between hypoxic v e n t i l a t o r y responses of awake (closed symbols) and hibernating (open symbols) ground s q u i r r e l s and hemoglobin-oxygen (Hb02) d i s s o c i a t i o n curves f o r blood at 37°C and 5°C. Note that at 5°C the Hb0 2 curve i s l e f t - s h i f t e d and the v e n t i l a t o r y response i s correspondingly reduced. Hb0 2 curves taken from Musacchia and Volkert (1971). 102 P n _ (mmHg) correspond to an alveolar P Q2 (pA02^ °^ about 7 t o r r , or approximately 40-50% saturation. The differ e n c e i n v e n t i l a t o r y responses at 3% 0 2 i s i n t e r e s t i n g i n that t h i s F j 0 2 would correspond to a PAC,2 °^ about 22 t o r r , or 80-90% saturation, and may indicate that Hb0 2 a f f i n i t y i s higher i n S. columbianus that i n S. l a t e r a l i s . However, i t i s d i f f i c u l t to draw d e f i n i t i v e conclusions without blood gas data and sp e c i e s - s p e c i f i c d i s s o c i a t i o n curves. That exposure to 1% 0 2 provoked arousal i n two of the golden-mantled ground s q u i r r e l s may suggest that hypoxic s e n s i t i v i t y i s higher i n t h i s species. Tahti (1975) also found that prolonged exposure to ins p i r e d 0 2 l e v e l s of l e s s than 3% would cause hibernating hedgehogs to arouse. The p o s s i b i l i t y that the two S. l a t e r a l i s that aroused from hibernation were not hibernating as deeply as those that d i d not cannot be ruled out, however, as s e n s i t i v i t y to external s t i m u l i i s known to increase as hibernation progresses, even i n the absence of any detectable changes i n Tg or HR (Landau and Dawe, 1958; Kr i s t o f f e r s s o n and Soivio, 1967; Pajunen, 1970; Twente and Twente, 1978). The decreased MR and the l e f t - s h i f t e d Hb0 2 d i s s o c i a t i o n curve greatly enhance the innate hypoxia tolerance of these two species of ground s q u i r r e l during hibernation (see Chapter 2). The hypoxic v e n t i l a t o r y responses recorded here suggest that changes i n PaQ2 P* ay 103 l i t t l e or no r o l e i n the control of v e n t i l a t i o n and breathing pattern i n S. l a t e r a l i s and S. columbianus during hibernation. The observation that hyperoxia has no e f f e c t on v e n t i l a t i o n i n hibernating ground s q u i r r e l s supports t h i s conclusion, since i t indicates that increasing P a 02 does not a l t e r any p r e v a i l i n g hypoxic v e n t i l a t o r y drive by decreasing peripheral chemoreceptor input. This i s of p a r t i c u l a r i n t e r e s t i n the case of S. l a t e r a l i s , as i t demonstrates that the l e v e l of P a 0 2 caused by i n s p i r a t i o n of 3% 0 2 i s probably not experienced during air-breathing, even at the end of the non-ventilatory period. Indeed, Steffen and Riedesel (1982) report an end-tidal P 0 2 of 90-95 t o r r at the beginning of the breathing period i n S. l a t e r a l i s . Thus despite the e f f e c t of 3% 0 2 on v e n t i l a t i o n i n t h i s species, changes i n P a 0 2 are u n l i k e l y to play a r o l e i n the regulation of CSR under normal conditions. Hypercapnia had a much greater e f f e c t on v e n t i l a t i o n than did hypoxia i n both S. l a t e r a l i s and S. columbianus. The pattern of v e n t i l a t o r y response to hypercapnia was the same i n both species of ground s q u i r r e l (Figures 17 and 18). V was increased by changes of both f and V T, though f increased much more than did V T (Figure 17). The increases i n f were due s o l e l y to decreases i n Tjjyp (Figure 18). In S. l a t e r a l i s , burst frequency increased i n proportion to the f a l l i n Tjjyp, but there was no change 104 i n the number of breaths taken per burst, or i n the timing of i n d i v i d u a l breaths (Figure 1 8 , Table 6 ) ; CSR was converted to continuous breathing at 3 - 5 % C 0 2 i n S. l a t e r a l i s (Figures 1 7 and 1 8 ) . In t h i s species, v a r i a b i l i t y of V T remained even when breathing had become continuous (Figure 1 4 ) . In addition, I found no i n t e r a c t i o n between changes i n F I 0 2 and F I C 0 2 on v e n t i l a t i o n i n hibernating ground s q u i r r e l s . The v e n t i l a t o r y responses to both hyperoxic and hypoxic hypercapnia were e s s e n t i a l l y the same as those f o r normoxic hypercapnia (Table 5 , Figure 1 9 ) . This f i n d i n g underlines the lack of a r o l e f o r hypoxia i n v e n t i l a t o r y control i n hibernating Columbian and golden-mantled ground s q u i r r e l s , since the hypercapnia-induced increase i n v e n t i l a t i o n would be expected to amplify the e f f e c t of a sustained decrease i n F I 0 2 by causing more rapid changes I N P A 0 2 -The hypercapnic v e n t i l a t o r y responses of hibernating S. l a t e r a l i s and S. columbianus are q u a l i t a t i v e l y s i m i l a r to those reported f o r the golden hamster and 1 3 - l i n e d ground s q u i r r e l (Lyman, 1 9 5 1 ) , the marmot (Endres and Taylor, 1 9 3 0 ) , and the hedgehog (Biorck et a l . , 1 9 5 6 ; Tahti, 1 9 7 5 ) . F J C Q 2 l e v e l s l e s s than 5 % caused a decrease i n Tjjyp and an increase i n f i n a l l of these species, but CSR was not converted to continuous breathing u n t i l F I C 0 2 exceeded 5 - 7 % (Lyman, 1 9 5 1 ; Biorck et a l . , 1 9 5 6 ; Tahti, 1 0 5 1975) . Hypercapnic s e n s i t i v i t y appears to be somewhat higher i n golden-mantled and Columbian ground s q u i r r e l s than i n e i t h e r the golden hamster or the marmot; however, i n part t h i s r e s u l t s from the lack of V T measurements i n a l l but one of these studies (Endres and Taylor, 1930). Even so, most of the increase i n V observed here was due to changes i n f and not to changes i n V T (Figure 17). At 5-7% C0 2, • V was increased by about 400% i n S. l a t e r a l i s and S. columbianus. owing to a 300% increase i n f and a 50% increase i n V T. An examination of the marmot data of Endres and Taylor (1930) reveals that V was increased by only about 200% at 7% C0 2. S i m i l a r l y , the hamster had increased i t s breathing frequency by about 200% at 5% C0 2 (Lyman, 1951). I f Lyman's data f o r the 13-lined ground s q u i r r e l respresents the response of the deeply hibernating animal, then t h i s species has markedly greater hypercapnic s e n s i t i v i t y than e i t h e r S. l a t e r a l i s or S. columbianus. For instance, he reported that exposure to 2-4% C0 2 caused f to r i s e from 1-5 breaths/minute to 15-23 breaths/minute, a f i v e - t o - t e n f o l d increase (Lyman, 1951). In most cases, however, the increase i n f was accompanied by an increase i n HR, which may indicate that h i s s q u i r r e l s were on the verge of arousing from hibernation; i n the one case where HR d i d not change, there was no e f f e c t of 2.5% C0 2 on f 106 (Lyman, 1951). That the v e n t i l a t o r y response to hypercapnia was achieved almost e n t i r e l y by decreases i n Tjjyp i n both s. l a t e r a l i s and S. columbianus suggests strongly that the non-ventilatory period i s the major c o n t r o l l e d v a r i a b l e i n both species, regardless of the intermittent breathing pattern. The observation that hypercapnia had no e f f e c t on T T 0 T , T j , or T E further implies that there was no a l t e r a t i o n of the integration of afferent information by the r e s p i r a t o r y centres. Rather, t h i s response pattern suggests that there i s some c r i t i c a l Paco2 o r P H that must be reached before the animal i s stimulated to breathe. Therefore, when F J C O 2 ^ s increased, the threshold Paco2 i s reached more quickly and Tjjyp becomes shorter. This scenario implies that breathing i s triggered to r e - e s t a b l i s h blood gas homeostasis or acid-base balance. In S. l a t e r a l i s . at l e a s t , t h i s appears to be true, since end-tidal P 0 2 increases and P^o2 decreases over the breathing period (Steffen and Riedesel, 1982). A c o r o l l a r y i s that S. columbianus requires only one or two breaths to do t h i s , while S. l a t e r a l i s requires several. Measurements of blood gases are required to v e r i f y t h i s p r e d i c t i o n . The e f f e c t s of hypoxia and hypercapnia on v e n t i l a t i o n and breathing pattern i n golden-mantled and Columbian ground s q u i r r e l s lead to the conclusion that, during hibernation, v e n t i l a t i o n i s con t r o l l e d mainly by changes i n 107 paC02 o r P H' w * t h changes i n P a 02 playing no r o l e . The two species have remarkably s i m i l a r o v e r a l l v e n t i l a t o r y responses and patterns of response to hypercapnia despite the d i f f e r e n c e i n t h e i r breathing patterns, c l e a r l y demonstrating that the apparent species d i f f e r e n c e i n intermittent breathing pattern cannot be explained on the basis of a difference i n s e n s i t i v i t y to resp i r a t o r y gases. Measurements of the v a r i a t i o n s of a r t e r i a l blood gases and pH during normal air-breathing as well as during hypoxic and hypercapnic exposure are required to support t h i s conclusion. In addition, nothing i s known about the ro l e s of lung and airway receptors nor pulmonary mechanics i n determining breathing pattern during hibernation. Studies of these variables w i l l undoubtedly provide greater i n s i g h t into the regulation of the intermittent breathing patterns displayed by hibernating animals. 108 CHAPTER FOUR GENERAL DISCUSSION During entrance into hibernation i n both the golden-mantled ground s q u i r r e l (Spermophilus l a t e r a l i s ) and the Columbian ground s q u i r r e l (S. columbianus), continuous breathing i s converted to intermittent breathing. In conjunction with the decrease i n body temperature (Tg) and metabolic rate (MR), o v e r a l l v e n t i l a t i o n (V) i s decreased tremendously i n hibernation compared to euthermia. The present study has shown that t h i s reduction i n v e n t i l a t i o n does not correspond to a loss of v e n t i l a t o r y c o n t r o l , as was thought by most previous investigators (Endres and Taylor, 1930; Biorck et a l . , 1956; Steffen and Riedesel, 1982). I t does, however, appear to involve an a l t e r a t i o n i n v e n t i l a t o r y c ontrol, insofar as v e n t i l a t o r y responses are not the same i n euthermia and hibernation. This i s true f o r both S. l a t e r a l i s and S. columbianus, regardless of the intermittent breathing pattern exhibited by the animal during hibernation at T A 5°C. Therefore the r e s u l t s do not support the hypothesis that the species difference i n breathing pattern can be explained on the basis of a differe n c e i n s e n s i t i v i t y to hypoxia and hypercapnia. When awake at T A 22°C, both species showed strong hypoxic v e n t i l a t o r y responses and comparatively blunted hypercapnic v e n t i l a t o r y responses (Figures 3 and 6). When 109 hibernating at T A 5°C, both showed greatly reduced hypoxic responses and comparatively elevated hypercapnic responses (Figures 15 and 17). These v e n t i l a t o r y responses suggest that changes i n P 0 2 play a greater r o l e i n v e n t i l a t o r y control i n awake ground s q u i r r e l s , whereas changes i n Pco2 o r P H a r e m o r e important i n hibernating animals. Moreover, the v e n t i l a t o r y responses of S. columbianus during p e r i o d i c arousal from hibernation, a l b e i t based on only three animals, seem to indicate that a seasonal a l t e r a t i o n of central v e n t i l a t o r y control has occurred. This may account, i n part, f o r the responses exhibited by the hibernating ground s q u i r r e l . Figure 21 i l l u s t r a t e s a comparison of the hypoxic and hypercapnic v e n t i l a t o r y responses of awake and hibernating ground s q u i r r e l s of both species. I t i s c l e a r that the increase i n V upon exposure to both hypoxic and hypercapnic gases i s g r e a t l y reduced i n hibernation. In part, t h i s i s due simply to the decrease i n r e s t i n g V and MR. When the v e n t i l a t o r y responses are expressed r e l a t i v e to the appropriate r e s t i n g MR of the animal, a very d i f f e r e n t p i c t u r e emerges. Figure 22 shows the r e l a t i v e hypoxic responses of both species while awake at T A 22°C and while hibernating at T A 5°C. Regardless of the manner of presentation, hypoxic v e n t i l a t o r y responses are obviously depressed dramatically during hibernation. This decrease i n hypoxic 110 Figure 21. Comparison of the v e n t i l a t o r y responses of awake (a) and hibernating (h) ground s q u i r r e l s to decreasing F I 0 2 and increasing F I C 0 2 . Each point respresents the mean + standard error f o r 5-7 animals. I l l Figure 22. Comparison of the e f f e c t s of decreasing F on V / V 0 2 / V t , and f expressed as percent change from control, i n awake and hibernating ground s q u i r r e l s . Symbols are as i n Figure 21. 112 112.0 s e n s i t i v i t y can be accounted f o r by the reduction i n MR and the increase i n hemoglobin-oxygen (Hb02) binding a f f i n i t y caused by the l e f t - s h i f t of the d i s s o c i a t i o n curve. I t i s not necessary to invoke any change i n the a c t i v i t y of peripheral chemoreceptors or i n the central integration of P o 2-induced changes i n afferent information from t h i s receptor group. In fac t , the fin d i n g that the hypoxic v e n t i l a t o r y response of S. columbianus during p e r i o d i c arousal i s i d e n t i c a l to that of the euthermic s q u i r r e l p r i o r to hibernation implies that there has been no long-term a l t e r a t i o n of hypoxic s e n s i t i v i t y . As discussed i n Chapter Three, P 5 0 i s reduced to 6-8 t o r r during hibernation (Clausen and Ersland, 1968; Musacchia and Volkert, 1971). Thus i t i s not too sur p r i s i n g that neither species of ground s q u i r r e l showed a v e n t i l a t o r y response to an apparent P Q 2 of about 22 t o r r ( F I 0 2 3%). However, the Hb0 2 d i s s o c i a t i o n curve of the hibernating animal i s also so steep that a drop i n PaQ2 o f even a few t o r r causes a substantial decrease i n a r t e r i a l 0 2 saturation ( S a 0 2 ) . Hypoxic s e n s i t i v i t y i s so low i n hibernation that when F I 0 2 * s decreased to 1% (7 torr) , cen t r a l hypoxic depression of metabolism may ensue before any peripheral stimulation of v e n t i l a t i o n can occur to o f f s e t the f a l l i n PaQ2 a n d sa02* On the other hand, r e l a t i v e hypercapnic responses are greater i n hibernation than i n euthermia (prior to 113 hibernation) (Figure 23). Some, though not a l l , of t h i s enhanced s e n s i t i v i t y appears to remain when the animal i s euthermic during p e r i o d i c arousal. In a l l euthermic animals, however, hypercapnic exposure increased v e n t i l a t o r y drive through an increase i n V T and decreases i n T T 0 T , T j , and Tj.' (Figure 6, Table 3). In hibernating animals, i t increased v e n t i l a t i o n p r i m a r i l y through a decrease i n Tjjyp (Figures 18). These differences i n the pattern of v e n t i l a t o r y response suggest that central v e n t i l a t o r y control has been altered i n some s t a t i c way during hibernation. What occurs during entrance into hibernation to account f o r these changes i n v e n t i l a t o r y control? One of the hallmarks of the beginning of the entry phase i s a decrease i n breathing frequency (Landau and Dawe, 1958; Lyman, 1958; Pajunen, 1970; Malan, 1982). This i s followed c l o s e l y by a drop i n metabolic rate, i n heart rate, and f i n a l l y , i n T B (Lyman, 1958; Strumwasser, 1960). The baseline MR of deep hibernation i s reached p r i o r to the l e v e l l i n g o f f of T B (Lyman, 1958; Pajunen, 1970), suggesting that something other than temperature i s a f f e c t i n g MR. Furthermore, calculated Q 1 0 values f o r the euthermia-to-hibernation decrease i n MR are often 3.0-3.5 (Malan, 1982). These observations strongly suggest that MR i s a c t i v e l y suppressed during entrance into hibernation. Hibernating animals maintain P c o 2 a n d P H cl°se to euthermic l e v e l s , despite the decrease i n T B (Stormont et 114 Figure 23. Comparison of the e f f e c t s of increasing F I C 0 2 on V7V 0 2\ V T / and f expressed as percent change from control i n awake and hibernating ground s q u i r r e l s . Symbols are as i n Figure 21. 114a 1140 a l . . 1939; Lyman and Hastings, 1951; Clausen, 1966; Kent and Peirce, 1967; Galbavy et a l . , 1972; Malan e£ a l . , 1973; Kreienbuhl et a l . , 1976). The maintenance of a r t e r i a l pH i n hibernation despite low T B constitutes a r e l a t i v e r e s p i r a t o r y a c i d o s i s compared to the euthermic state (Malan, 1980, 1982).Chronic acidosis has been shown to depress the threshold f o r shivering thermogenesis (probably by i n h i b i t i n g neurons i n the pre-optic area of the hypothalamus) (Schaefer et a l . , 1975; Schaefer and Wunnenberg, 1976; Jennings, 1979), as well as to i n h i b i t non-shivering thermogenesis and several key metabolic pathways (e.g. g l y c o l y s i s ) (Malan, 1980, 1982). The development of r e l a t i v e acidosis during entrance i n t o hibernation has been proposed to suppress MR beyond the l e v e l predicted by the f a l l i n T B (Malan, 1980, 1982). Since C0 2 s o l u b i l i t y increases as temperature f a l l s , from 0.03 mmol/litre/torr at 37°C to 0.07 mmol/litre/torr at 6°C (Malan, 1982), the maintenance of constant P C 02 and pH i n hibernation requires the addition of a large amount of C0 2 to the blood. C0 2 retention could be achieved during entrance simply by a s h i f t i n the r a t i o of v e n t i l a t o r y C0 2 elimination to metabolic C0 2 production ( V / V C 0 2 ) , and would be easiest early i n the entry phase, when T B and MR are s t i l l high. Further, a c i d o t i c suppression could be reversed r a p i d l y p r i o r to arousal simply by an increase i n v e n t i l a t i o n . These changes i n the 115 V/V C 02 r a t i o demand changes i n v e n t i l a t o r y c o n t r o l . During entrance, active C0 2 retention v i a a s h i f t i n v e n t i l a t o r y control must involve an increase i n the response threshold (decrease i n s e n s i t i v i t y ) , so that f o r any P C 0 2 , the animal i s hypoventilating r e l a t i v e to the awake state. This i s known to occur at the onset of sleep i n non-hibernating mammals, where i t precedes the declines i n T B and MR ( P h i l l i p s o n , 1978). Due to the d i f f i c u l t y of studying v e n t i l a t i o n during the t r a n s i t i o n a l phases of entry and arousal, there i s no d i r e c t evidence to indicate that active C0 2 retention occurs during entrance into hibernation. Snapp and H e l l e r (1981) reported a b r i e f t r a n s i t o r y change i n respi r a t o r y quotient (RQ) during entrance i n S. l a t e r a l i s ; on the basis of t h i s observation, these authors concluded that though res p i r a t o r y acidosis might i n h i b i t thermoregulation, i t d i d not a c t i v e l y suppress MR. More recently, B i c k l e r (1984) has shown that C0 2 retention accompanies the decline i n Tg during the onset of both sleep and torpor i n the heterothermic round-tailed ground s q u i r r e l (S. tereticaudus), but does not occur when T B f a l l s i n the awake animal. In addition, i n t h i s study Q j 0 was much higher i n the t o r p i d s q u i r r e l than i n the awake animal at low Tg (Bickler, 1984). These data support the proposal that C0 2 retention and respi r a t o r y acidosis a c t i v e l y suppress MR during entrance into torpor and 116 hibernation. In contrast to the t r a n s i t o r y changes i n RQ noted by Snapp and H e l l e r (1981), B i c k l e r (1984) observed a prolonged period of C0 2 retention (two hours) during entry i n t o torpor of S. tereticaudus. This f i n d i n g suggests that there i s a gradual change i n v e n t i l a t o r y control during entrance int o torpor i n t h i s species. The changes i n breathing pattern during entrance i n S. l a t e r a l i s suggest, i n contrast, that v e n t i l a t o r y control changes abruptly. In what appears to be early entrance, short non-ventilatory periods appear suddenly amidst the breaths; as entrance progresses, Tjjyp gets longer u n t i l the CSR pattern c h a r a c t e r i s t i c of deep hibernation i s f u l l y developed (Figure 24). S i m i l a r l y , during arousal, the return to continuous breathing occurs very r a p i d l y . A p a r a l l e l s e r i e s of events occurs i n S. columbianus. except that the Tjjyp are, at a l l times, separated by only one or two breaths (personal observation). With regard to the arousal phase, t h i s species d i f f e r s from another that shows a single-breath intermittent breathing pattern during deep hibernation (S. r i c h a r d s o n i i ) . In S. r i c h a r d s o n i i . a pattern s i m i l a r to CSR occurs t r a n s i e n t l y during arousal (personal observation). Thus despite the i d e n t i c a l intermittent breathing patterns of hibernating S. columbianus and S. r i c h a r d s o n i i . there are apparently differences i n v e n t i l a t o r y control during arousal; t h i s may also hold f o r 117 Figure 24. Changes i n breathing pattern during entrance, hibernation, and arousal i n the golden-mantled ground s q u i r r e l . 118 e n t r a n c e g o l d e n m a n t l e d g r o u n d s q u i r r e l i i 1 min entrance. The r e l a t i v e l y increased hypercapnic responses observed i n both S. l a t e r a l i s and S. columbianus during hibernation and i n S. columbianus during periodic arousal would seem to contradict the decrease i n C0 2 s e n s i t i v i t y required for a c t i v e C0 2 retention and respiratory a c i d o s i s . I t i s possible, i n S . columbianus at l e a s t , however, that a seasonal s h i f t i n C0 2 s e n s i t i v i t y occurred s h o r t l y before the f i r s t entry into hibernation. While the animal remained euthermic, v e n t i l a t i o n would be matched to V 0 2 and V C 0 2 , since hypoxic s e n s i t i v i t y appears to be the more prominent fa c t o r i n v e n t i l a t o r y control i n the awake state. The proposed down-shift i n v e n t i l a t o r y control and uncoupling of V and V C 0 2 might s t i l l occur, and would s t i l l produce a r e l a t i v e r e s p i r a t o r y a c i d o s i s . This putative s h i f t i n v e n t i l a t o r y control would have been undetected i n t h i s study, since the animals were not tested j u s t p r i o r to hibernation. I t seems reasonable to assume that S . l a t e r a l i s might show a s i m i l a r pattern, since the v e n t i l a t o r y responses of t h i s species were the same as those of S. columbianus i n both euthermia and hibernation. The species difference i n intermittent breathing pattern displayed by hibernating S . l a t e r a l i s and S . columbianus i s not related to v e n t i l a t o r y responses to hypoxia or hypercapnia. Whether i t r e s u l t s from a difference i n the input from pulmonary receptors i s not known, since 119 these have not been examined i n any hibernating animal. More probably, during hibernation there i s a species difference i n the c e n t r a l nervous integration of afferent information. The observation of Hammel et a l . (1968) of temperature-dependent breathing patterns i n S. l a t e r a l i s i s i n t e r e s t i n g i n t h i s respect, since i t suggests that temperature a f f e c t s the integration of information from sensory receptor groups. This could occur d i r e c t l y , v i a changes i n neuronal transmission, as appears to be the case i n hypothermic cats (Kiley et a l . , 1984). Conversely i t might occur i n d i r e c t l y , by e f f e c t s on factors such as blood acid-base status or lung and chest-wall compliances. Whether S. columbianus shows a s i m i l a r e f f e c t of T A on intermittent breathing pattern and what changes i n v e n t i l a t o r y control accompany these t r a n s i t i o n s i n intermittent breathing pattern remain e x c i t i n g prospects f o r future research. In conclusion, t h i s study has shown that entrance into hibernation i n golden-mantled and Columbian ground s q u i r r e l s corresponds to a t r a n s i t i o n from continuous breathing to intermittent breathing, to a marked reduction i n hypoxic s e n s i t i v i t y , and to a r e l a t i v e increase i n hypercapnic s e n s i t i v i t y . Further, i t has shown that control of breathing during hibernation d i f f e r s dramatically from that of euthermia, as the non-ventilatory period, rather than the breath, becomes the major c o n t r o l l e d v a r i a b l e of the 120 breathing pattern. Most importantly, the r e s u l t s show c l e a r l y that these patterns of v e n t i l a t o r y change are i d e n t i c a l i n both species. Thus a difference i n v e n t i l a t o r y s e n s i t i v i t y to respiratory gases cannot be the basis for the presence of the two d i f f e r e n t intermittent breathing patterns displayed by these two species of ground s q u i r r e l during hibernation. 121 LITERATURE CITED Ar, A., R. A r i e l i , and A. Shkolnik. 1977. Blood gas properties and function i n the f o s s o r i a l mole r a t under normal and hypoxic-hypercapnic atmospheric conditions. Respir. Physiol. 30: 201-218. A r i e l i , R. 1979. The atmospheric environment of the f o s s o r i a l mole r a t (Spalax ehrenberai): e f f e c t s of season, s o i l texture, r a i n , temperature, and a c t i v i t y . Comp. Biochem. Physiol. 63A: 569-575. A r i e l i , R. and A. Ar. 1979. V e n t i l a t i o n of a f o s s o r i a l mammal (Spalax ehrenberqi) i n hypoxic and hypercapnic conditions J . Appl. Physiol. 47: 1011-1017. Bartels, H., R. SChmelzle, and S. U l r i c h . 1969. Comparative studies of the respiratory function of mammalian blood V. Insectivora: shrew, mole, and nonhibernating and hibernating hedgehog. Respir. Phsyiol. 7:278-286. Baudinette, R.V. 1974. Physiological correlates of burrow gas conditions i n the C a l i f o r n i a ground s q u i r r e l . Comp. Biochem. Physiol. 48A: 733-743. Benedict, F.G. and R.C. Lee. 1938. Hibernation and Marmot Physiology. Carnegie I n s t i t u t e of Washington publication. B i c k l e r , P.E. 1984. CO, balance of a heterothermic rodent: comparison of sleep, torpor, and awake states. Am. J . Physiol. 246: R49-R55. Biorck, G., B. Johansson, and H. Schmid. 1956. Reactions of hedgehogs, hibernating and non-hibernating, to the inha l a t i o n of oxygen, carbon dioxide, and nitrogen. Acta. Physiol. Scand. 37: 71-83. Birchard, G.F., D.F. Boggs, and S.M. Tenney. 1984. E f f e c t of pe r i n a t a l hypercapnia on the adult v e n t i l a t o r y response to carbon dioxide. Respir. Physiol. 57: 341-347. Boggs, D.F. and D.L. Kilgore, J r . 1983. Ve n t i l a t o r y responses of the burrowing owl and bobwhite to hypercarbia and hypoxia. J . Comp. Physiol. 149: 527-533. Boggs, D.F., D.L. Kilgore, and G.F. Birchard. 1984. Minireview: re s p i r a t o r y physiology of burrowing mammals and birds. Comp. Biochem. Physiol. 77A: 1-7. Bowes, G., E.R. Townsend, L. Kozar, S. Bromley, and E.A. P h i l l i p s o n . 1981. E f f e c t of c a r o t i d body denervation on 122 arousal response to hypoxia i n sleeping dogs. J . Appl. Physiol. 51: 40-45. Bullard, R.W. and F.R. Meyer. 1966. Relationship of hypoxic heart rate response to ambient temperature. J . Appl. Physiol. 21(3): 999-1003. Chapin, J.L. 1954. Ven t i l a t o r y response of the unrestrained and unanaesthetized hamster to C0 2. Am. J . Physiol. 179: 146-148. Chappell, M.A. 1985. E f f e c t s of ambient temperature and a l t i t u d e on v e n t i l a t i o n and gas exhange i n deer mice (Peromyscus  maniculatus). J . Comp. Physiol. B 155: 751-758. Chapman, R.C. and A.F. Bennett. 1975. Physiological correlates of burrowing i n rodents. Comp. Biochem. Physiol. 51A: 599-603. Cherniack, N.S. 1981. Respiratory dysrhythmias during sleep. N. Engl. J . Med. 305(6): 325-330. Cherniack, N.S. and G.S. Longobardo. 1981. The chemical control of r e s p i r a t i o n . Ann. Biomed. 9: 395-407. Cherniack, N.S. and G.S. Longobardo. 1983. Mathematical model of peri o d i c breathing during sleep. In: Modelling and  Control of Breathincr. eds. B.J. Whipp and D.M. Wiberg, E l s e v i e r Science Publishing Co., Inc. Clausen, G. 1966. Acid-base balance i n the hedgehog Erinaceus  europaeus L. during hibernation, hypothermia, cooling, and rewarming. Acta u n i v e r s i t a t i s bergensis: series mathematica. Norwegian u n i v e r s i t i e s press. Bergen, Oslo. Clausen, G. and A. Ersland. 1968. The respir a t o r y properties of the blood of the hibernating hedgehog Erinaceus europaeus Respir. Physiol. 5: 221-233. Cragg, P.A. and D.B. Drysdale. 1983. Interaction of hypoxia and hypercapnia on v e n t i l a t i o n , t i d a l volume, and respiratory frequency i n the anaesthetized r a t . J . Physiol. 341: 477-493. Darden, T.R. 1972. Respiratory adaptations of a f o s s o r i a l mammal the pocket gopher (Thomomys bottae). J . Comp. Physiol. 78: 121-137. Dejours, P. 1981. P r i n c i p l e s of Comparative Respiratory Physiology. 2nd revised e d i t i o n . Amsterdam, New York, Oxford, Elsevier/North-Holland Biomedical Press. 256pp. 123 Dempsey, J.A. and H.V. Forster. 1982. Mediation of ven t i l a t o r y adaptations. Physiol. Rev. 62: 262-346. Dowell, A.R., C E . Buckley I I I , R. Cohen, R.E. Whalen, and H.O. Sieker. 1971. Cheyne-Stokes r e s p i r a t i o n : a review of c l i n i c a l manifestations and a c r i t i q u e of physiological mechanisms. Arch. Int. Med. 127: 712-726. Drorbaugh, J.E. and W.O. Fenn. 1955. A barometric method for measuring v e n t i l a t i o n i n newborn infants. Pediatrics 16: 81-87. Endres, G. and H. Taylor. 1930. Observation on c e r t a i n physio-l o g i c a l processes of the marmot. I I . The re s p i r a t i o n . Proc. Roy. Soc. (London) Ser. B. 107: 231-240. Epstein, M.A.F. and R.A. Epstein. 1978. A t h e o r e t i c a l analysis of the barometric method f o r measurement of t i d a l volume. Respir. Physiol. 32: 105-120. Epstein, R.A., M.A.F. Epstein, G.G. Haddad, and R.B. Mellin s . 1980. P r a c t i c a l implementation of the barometric method for measurement of t i d a l volume. J . Appl. Physiol. 49(6): 1107-1115. F a l e s c h i n i , R.J. and B.K. Whitten. 1975. Comparative hypoxic tolerance i n the Sciuridae. Comp. Biochem. Physiol. 52: 217-222. Fleming, P.J., M.R. Levine, A.L. Goncalves and S. Woollard. 1983. Barometric plethysmograph: advantages and l i m i t a t i o n s i n recording infant r e s p i r a t i o n . J . Appl. Physiol. 55: 1924-1931. Florant, G.L., B.M. Turner, and H.C. H e l l e r . 1978. Temperature regulation during wakefulness, sleep, and hibernation i n marmots. Am. J . Physiol. 235: R82-R88. Galbavy, E.J., J.A. Panuska, T.F. Albert, and M.M. Orlando. 1972. Blood gases of woodchucks at reduced body temperatures. J . Mammal. 53: 919-921. Glotzbach, S.F. and H.C. H e l l e r . 1978. Central nervous regulation of body temperature during sleep. Science 194: 537-539. Goodrich, C.A. 1973. Acid-base balance i n euthermic and hibernating marmots. Am. J . Physiol. 224: 1185-1189. Guilleminault, C., A. T i l k i a n , and W.C. Dement. 1976. The sleep apnea syndromes. Ann. Rev. Med. 27: 4 65-484. 124 Guyton, A.C., J.W. Crowell, and J.W. Moore. 1957. Basic o s c i l l a t i n g mechanism of Cheyne-Stokes r e s p i r a t i o n . Am. J . Physiol. 187: 395-398. H a l l , F.G. 1965. Minimal u t i l i z a b l e oxygen and the oxygen d i s s o c i a t i o n curve of blood of rodents. J . Appl. Physiol. 21: 375-378. H a l l , Marshall. 1836. Hibernation. In: Todd 1s Cyclopaedia of  Anatomy and Physiology. Vol. I I . p. 769. Hammel, H.T., T.J. Dawson, R.M. Abrams, and H.T. Anderson. 1968. Total c a l o r i m e t r i c measurements on C i t e l l u s l a t e r a l i s i n hibernation. Physiol. Zool. 41 : 341-357. Harkness, D.R., S. Roth, and P. Goldman. 1974. Studies on the red blood c e l l oxygen a f f i n i t y and 2,3-diphosphoglyceric ac i d i n the hibernating woodchuck (Marmota monax). Comp. Biochem. Physiol. 48A: 591-599. Hayward, J.S. 1966. Abnormal concentrations of respi r a t o r y gases i n r abbit burrows. J . Mammal. 47(4): 723-724. Hedemark, L.L. and R.S. Kronenberg. 1982. Ve n t i l a t o r y and heart rate responses to hypoxia and hypercapnia during sleep i n adults. J . Appl. Physiol. 53: 307-312. Hemingway, A. Measurement of airway resistance with the body  plethysmography C C . Thomas, S p r i n g f i e l d , IL. 1973. 104pp. Hel l e r , H.C. 1979. Hibernation: neural aspects. Ann. Rev. Physiol. 41: 305-321. Hel l e r , H.C, G.W. C o l l i v e r , and J . Beard. 1977. Thermoregulation during entrance into hibernation. Pflugers Archiv. 369: 55-59. He l l e r , H.C, G.L. Florant, S.F. Glotzbach, J.M. Walker, and R.J. Berger. 1978. Sleep and torpor: homologous adaptations f o r energy conservation. In: Dormancy and  Developmental Arrest: Experimental Analysis i n Plants  and Animals, ed. M.E. Clutter, New York, Academic Press, pp. 269-296. H e l l e r , H.C. and S.F. Glotzbach. 1977. Thermoregulation during sleep and hibernation. In: Environmental Physiology I I . Int. Rev. Physiol. 15: 147-188. He l l e r , H.C, J.M. Walker, G.L. Florant, S.F. Glotzbach, and R.J. Berger. 1978. Sleep and hibernation: 125 f e l e c t r o p h y s i o l o g i c a l and thermoregulatory homologies. In: Strategies i n Cold pp. 225-265. Hiestand, W.A., W.T. Rockhold, F.W. Stemler, D.E. Stullken, and J.E. Wiebers. 1957. The comparative hypoxic resistance of hibernators and non-hibernators. Physiol. Zool. 23(4): 264-268. Holloway, D.A. and A.G. Heath. 1984. Ve n t i l a t o r y changes i n the golden hamster, Mesocricetus auratus. compared with the laboratory r a t , Rattus norvegicus. during hypercapnia and/or hypoxia. Comp. Biochem. Physiol. 77A: 267-273. Hoover, W.H., P.J. Young, M.S. Sawyer, and W.P. Apgar. 1970. Ovine p h y s i o l o g i c a l responses to elevated ambient carbon dioxide. J . Appl. Physiol. 29: 32-35. Hudson, J.W. 1978. Shallow, d a i l y torpor: a thermoregulatory adaptation. In: Strategies i n Cold, pp. 67-108. Hudson, J.W. and D.R. Deavers. 1973. Metabolism, pulmocutaneous water loss and r e s p i r a t i o n of eight species of ground s q u i r r e l s from d i f f e r e n t environments. Comp. Biochem. Physiol. 45A: 69-100. Jacky, J.P. 1978. A plethysmograph for long-term measurements of v e n t i l a t i o n i n unrestrained animals. J . Appl. Physiol. 45: 644-647. Jacky, J.P. 1980. Barometric measurement of t i d a l volume: e f f e c t s of pattern and nasal temperature. J . Appl. Physiol. 49: 319-325. Javaheri, S., E.C. Lucey, and G.L. Studer. 1980. Ve n t i l a t o r y response to carbon dioxide breathing i n unanesthetized unrestrained hamsters. C l i n . Res. 28: 529A. Jennings, D.B. 1979. Body temperature and v e n t i l a t o r y response to CO, during chronic v e n t i l a t o r y a c i d o s i s . J . Appl. Physiol. 46: 491-497. Jennings, D.B. and J.S.D. Davidson. 1984. Acid-base and v e n t i l a t o r y adaptation i n conscious dogs during chronic hypercapnia. Respir. Physiol. 58: 377-393. Kent, K.M. and E.C. Peirce I I . 1967. Acid-base c h a r a c t e r i s t i c s of hibernating animals. J . Appl. Physiol. 23: 336-340. Khoo, M.C.K., R.E. Kronauer, K.P. Strohl, and A.S. Slutsky. 1982. Factors inducing periodic breathing i n humans: a general model. J . Appl. Physiol. 53(3): 644-659. 126 K i l e y , J.P. r F.L. Eldridge, and D.E. Millhorn. 1984. The e f f e c t of hypothermia on central neural control of r e s p i r a t i o n . Respir. Physiol. 58: 295-312. Kreienbuhl, G., J . Strittmatter, and E. Ayim. 1976. Blood gas analyses of hibernating hamsters and dormice. Pflugers Archiv. 366: 167-172. K r i s t o f f e r s s o n , R. and A. Soivio. 1964. Hibernation i n the hedgehog (Erinaceus europaeus L.). Changes of respiratory pattern, heart rate, and body temperature i n response to gradually decreasing or increasing ambient temperature. Ann. Acad. S c i . Fenn. Series A. IV B i o l . 82: 3-17. K r i s t o f f e r s s o n , R. and A. Soivio. 1966. Duration of hypothermia periods and type of r e s p i r a t i o n i n the hibernating golden hamster, Mesocricetus auratus Waterhouse. Ann. Zool. Fenn. 3: 66-67. Kr i s t o f f e r s s o n , R. and A. Soivio. 1967. Studies on the period-i c i t y of hibernation i n the hedgehog Erinaceus europaeus L. I I . Changes i n respiratory rhythm, heart rate, and body temperature at the onset of spontaneous and induced arousals. Ann. Zool. Fenn. 4: 595-597. L a h i r i , S., K. Maret, and M.G. Sherpa. 1983. Evidence of high a l t i t u d e sleep apnea on v e n t i l a t o r y s e n s i t i v i t y to hypoxia. Respir. Physiol. 52: 281-301. L a h i r i , S. and R. Gelfand. 1981. Mechanisms of acute v e n t i l a t o r y responses. In: Regulation of Breathing I I . ed. T. Hornbein. pp.773-821. L a i , Y-L., Y. Tsuya, and J . Hildebrandt. 1978. V e n t i l a t o r y responses to acute C0 2 exposure i n the r a t . J . Appl. Physiol. 45: 611-618. Landau, B.R. and A.R. Dawe. 1958. Respiration i n the hibernation of the t h i r t e e n - l i n e d ground s q u i r r e l . Am. J . Physiol. 194: 75-82. Lange, R.L. and H.H. Hecht. 1962. The mechanism of Cheyne-Stokes r e s p i r a t i o n . J . C l i n . Invest. 41(1): 42-52. Lechner, A.J. 1976. Respiratory adaptations i n burrowing pocket gophers from sea l e v e l and high a l t i t u d e . J . Appl. Physiol. 41: 168-173. Lechner, A.J. 1985. Pulmonary design i n a microchiropteran bat ( P i p i s t r e l l u s subflavus) during hibernation. Respir. Physiol. 59: 301-312. 127 L e d l i e , J.F., S.G. Kelsen, N.S. Cherniack, and A.P. Fishman. 1981. E f f e c t s of hypercapnia and hypoxia on phrenic nerve a c t i v i t y and respiratory timing. J . Appl. Physiol. 51: 732-738. L e i t h , D.E. and J . Mead. P r i n c i p l e s of Body Plethysmography. National Heart Lung I n s t i t u t e , Bethesda, MD. 1974. Leitner, L-M., and A. Malan. 1973. Possible r o l e of the a r t e r i a l chemoreceptors i n the v e n t i l a t o r y responses of the anaesthetized marmot to changes i n ins p i r e d 0, p a r t i a l pressure and C0 2 p a r t i a l pressure. Comp. Biochem. Physiol. 45A: 953-959. Longobardo, G.S., B. Gothe, M.D. Goldman, and N.S. Cherniack. 1982. Sleep apnea considered as a control system i n s t a b i l i t y . Respir. Physiol. 50: 311-333. Lyman, C P . 1951. E f f e c t of increased C0 2 on r e s p i r a t i o n and heart rate of hibernating hamsters and ground s q u i r r e l s . Am. J . Physiol. 167: 638-643. Lyman, C P . 1958. Oxygen consumption, body temperature, and heart rate of woodchucks entering hibernation. Am. J . Physiol. 194: 83-91. Lyman, C P . 1982. Why bother to hibernate? In: Hibernation and Torpor i n Mammals and Birds, eds. C P . Lyman, J.S. W i l l i s A. Malan, and L.C.H. Wang, New York, Academic Press, pp. 1-36. Lyman, C P . and P.O. C h a t f i e l d . 1955. Physiology of hibernation i n mammals. Physiol. Rev. 35: 403-425. Lyman, C P . and A.B. Hastings. 1951. Total CO,, plasma pH, and PC02 °^ hamsters and ground s q u i r r e l s during hibernation. Am. J . Physiol. 167: 633-637. Maclean, G.S. 1981. Torpor patterns and microenvironment of the eastern chipmunk, Tamias s t r i a t u s . J . Mammal. 62: 64-73. Maclean, G.S. 1981. Factors influencing the composition of res p i r a t o r y gases i n mammal burrows. Comp. Biochem. Physiol. 69A: 373-380. McNab, B.K. 1966. The metabolism of f o s s o r i a l rodents: a study of convergence. Ecology 47: 712-733. Malan, A. 1973. V e n t i l a t i o n measured by body plethysmography i n hibernating mammals and poikilotherms. Respir. Physiol. 17: 32-44. 128 Malan, A. 1980. Enzyme regulation, metabolic rate, and acid-base state i n hibernation. In: Animals and Environmental  Fitness, ed. R. G i l l e s , Oxford and New York, Pergamon, pp. 487-501. Malan, A. 1982. Respiration and acid-base state i n hibernation. In: Hibernation and Torpor i n Mammals and Birds, eds. C P . Lyman, J.S. W i l l i s , A. Malan, and L.C.H. Wang. New York, Academic Press, pp. 273-282. Malan, A., H. Arens, and A. Waechter. 1973. Pulmonary r e s p i r a t i o n and acid-base state i n hibernation marmots and hamsters. Respir. Physiol. 17: 45-61. Morrison, P.R. 1960. Some i n t e r r e l a t i o n s between weight and hibernation function. B u l l . Mus. Comp. Zool. 124: 75-91. Musacchia, X.J. and W.A. Volkert. 1971. Blood gases i n hibernating and active ground s q u i r r e l s : Hb0 2 a f f i n i t y at 6 and 38°C Am. J . Physiol. 221: 128-130. Netick, A., J . Orem, and W. Dement. 1977. Neuronal a c t i v i t y s p e c i f i c to REM sleep and i t s r e l a t i o n s h i p to breathing. Brain Res. 120: 197-207. Neubauer, J.A., T.V. Santiago, and N.H. Edelman. 1981. Hypoxic arousal i n i n t a c t and c a r o t i d chemodenervated sleeping cats. J . Appl. Physiol. 51: 1294-1299. Orem, J . 1980. Medullary respiratory neuron a c t i v i t y : r e l a t i o n -ship to ton i c and phasic REM sleep. J . Appl. Physiol. 48: 54-65. Orem, J . , A. Netick, and W.C Dement. 1977. Breathing during sleep and wakefulness i n the cat. Respir. Physiol. 30: 265-289. Pajunen, I. 1970. Body temperature, heart rate, breathing pattern, weight l o s s , and p e r i o d i c i t y of hibernation i n the Finnish garden dormouse, Eliomys quercinus L. Ann. Zool. Fenn. 7: 251-266. Pajunen, I. 1974. Body temperature, heart rate, breathing pattern, weight l o s s , and p e r i o d i c i t y of hibernation i n the French garden dormouse, Eliomys quercinus at 4.2 + 0.5°C Ann. Zool. Fenn. 11: 107-119. Pappenheimer, J.R. 1977. Sleep and r e s p i r a t i o n of rats during hypoxia. J . Physiol. 266: 191-207. 129 Pembrey, M.S. and A.G. P i t t s . 1899. The r e l a t i o n between the in t e r n a l temperature and the respiratory movements of hibernating animals. J . Physiol. (London) 24: 305-316. Pengelley, E.T. and K.C. Fisher. 1961. Rhythmical arousal from hibernation i n the golden-mantled ground s q u i r r e l C i t e l l u s l a t e r a l i s tescorum. Can. J . Zool. 39: 105-120. P h i l l i p s o n , E.A. 1978. Respiratory adaptations i n sleep. Ann. Rev. Physiol. 40: 133-156. P h i l l i p s o n , E.A., E. Murphy, and L.F. Kozar. 1976. Regulation of r e s p i r a t i o n i n sleeping dogs. J . Appl. Physiol. 40: 688-693. Rasmussen, A.T. 1916. Theories of hibernation. Am. Nat u r a l i s t 50: 609-625. Remmers, J.E. 1981. E f f e c t s of sleep on control of breathing. I n t ' l . Review of Physiol. Respiration Physiology I I I . v o l . 23. pp. 111-147. Schaefer, K.E., A.A. Messier, C. Morgan, and G.T. Baker. 1975. E f f e c t of chronic hypercapnia on body temperature regulation. J . Appl. Physiol. 38: 900-906. Schaefer, K. and W. Wunnenberg. 1976. Threshold temperatures for shivering i n acute and chronic hypercapnia. J . Appl. Physiol. 41: 67-70. Scheck, S.H. and E.D. Fleharty. 1980. Subterranean behaviour of the adult t h i r t e e n - l i n e d ground s q u i r r e l , Spermophilus  tridecemlineatus. American Midland N a t u r a l i s t 103: 191-195. Schlenker, E.H. 1985. V e n t i l a t i o n and metabolism of the Djungarian hamster (Phodopus sunqorus) and the albino mouse. Comp. Biochem. Physiol. 82A(2): 293-295. Schlenker, E.H. and C F . Herreid. 1981. The e f f e c t of low level s of carbon dioxide on metabolism of Mus musculus. Comp. Biochem. Physiol. 68A: 673-676. Schmid, W.D. 1976. Temperature gradients i n the nasal passage of some small mammals. Comp. Biochem. Physiol. 54A: 304-308. Snapp, B.D. and H.C He l l e r . 1981. Suppression of metabolism during hibernation i n ground s q u i r r e l s ( C i t e l l u s  l a t e r a l i s ) . Physiol. Zool. 54: 297-307. 130 Snedecor, G.W. and W.G. Cochran. 1980. S t a t i s t i c a l Methods. 7th e d i t i o n . Ames, Iowa, State University Press. Soholt, L.F., M.K. Yousef, and D.B. D i l l . 1973. Responses of Merriam's kangaroo rats, Dipodomvs merriami. to various l e v e l s of carbon dioxide concentration. Comp. Biochem. Physiol. 45A: 455-462. Spallanzani, L. Memoirs sur l a Respiration, par Senebier. Geneva, 1803. Stahl, W.R. 1967. Scaling of respiratory variables i n mammals. J . Appl. Physiol. 22: 453-460. Steffen, J.M. and M.L. Reidesel. 1982. Pulmonary v e n t i l a t i o n and cardiac a c t i v i t y i n hibernating and arousing golden-mantled ground s q u i r r e l s (Spermophilus l a t e r a l i s ) . Cryobiology 19: 83-91. Stormont, R.T., M.A. Foster, and C. P f e i f f e r . 1939. Plasma pH, C0 2 content of the blood, and "tissue gas" tensions during hibernation. Proc. Soc. Exp. B i o l . Med. 42: 56-59. Strumwasser, F. 1959. Thermoregulatory, brain, and behavioural mechanisms during entrance into hibernation i n the s q u i r r e l , C i t e l l u s beecheyi. Am. J . Physiol. 196: 15-22. Strumwasser, F. 1959. Regulatory mechanisms, brain a c t i v i t y , and behaviour during deep hibernation i n the s q u i r r e l , C i t e l l u s beecheyi. Am. J . Physiol. 196: 23-30. Studier, E.H. and J.W. Procter. 1971. Respiratory gases i n burrows of Spermophilus tridecemlineatus . J . Mammal. 52: 631-633. S u l l i v a n , C E . 1981. Breathing i n Sleep. In: Physiology i n Sleep eds. J . Orem and CD. Barnes. New York, Academic Press, pp. 213-272. Tahti, H. 1975. E f f e c t s of changes i n C0 2 and 0 2 concentrations i n the inspired gas on r e s p i r a t i o n i n the hibernating hedgehog (Erinaceus europaeus L.). Ann. Zool. Fenn. 12: 183-187. Tahti, H. and A. Soivio. 1975. Blood gas concentrations, a c i d -base balance, and blood pressure i n hedgehogs i n the act i v e state and i n hibernation with p e r i o d i c r e s p i r a t i o n . Ann. Zool. Fenn. 12: 188-192. Tahti, H., M. Nikinmaa, and A. Soivio. 1981. Cheyne-Stokes breathing pattern as an adaptation to deep 131 hibernation hypothermia. Cryobiology 18: 92. Twente, J.W. and J.A. Twente. 1978. Autonomic regulation of hibernation by C i t e l l u s and Eptesicus. In: Strategies i n Cold, Natural T o r p i d i t y and Thermogenesis. eds. L.C.H. Wang and J.W. Hudson, New York, Academic Press, pp. 327-373. Vogel, S., C P . E l l i n g t o n , J r . , and D.L. Kilgore, J r . 1973. Wind-induced v e n t i l a t i o n of the burrow of the p r a i r i e dog Cynomys ludovicianus. J . Comp. Physiol. 85: 1-14. Waggener, T.B., P.J. B r u s i l , R.E. Kronauer, R.A. Gabel, and G.F. Inbar. 1984. Strength and cycle time of high-altitude v e n t i l a t o r y patterns i n unacclimatized humans. J . Appl. Physiol. 56: 576-581. Walker, J.M., S.F. Glotzbach, R.J. Berger, and H.C Heller.1977. Sleep and hibernation i n ground s q u i r r e l s ( C i t e l l u s spp): e l e c t r o p h y s i o l o g i c a l observations. Am. J . Physiol. 233: R213-R221. Walker, B.R., E.M. Adams, and N.F. Voelkel. 1985. Ventilatory responses of hamsters and rats to hypoxia and hypercapnia. J . Appl. Physiol. 59(6): 1955-1960. Wang, L.C.H. 1978. Energetic and f i e l d aspects of mammalian torpor: the Richardson's ground s q u i r r e l . In: Strategies i n Cold. Natural T o r p i d i t y and Thermogenesis. eds. L.C.H. Wang and J.W. Hudson. New York, Academic Press, pp. 109-145. Weil, J.V., M.H. Kryger, and CH. Scoggin. 1978. Sleep and breathing at high a l t i t u d e . In: Sleep Apnea Syndromes. eds. C. Guilleminault and W.C Dement. New York, Alan R. L i s s , Ltd. pp. 119-136. Williams, D.D. and R.L. Rausch. 1973. Seasonal C0 2 and 0 2 concentrations i n the dens of hibernating mammals Sciuridae. Comp. Biochem. Physiol. 44A: 1227-1235. Wit, L.C and J.W. Twente. 1983. The e f f e c t s of hibernation stress on the heart rate and metabolic rate of C i t e l l u s l a t e r a l i s . Comp. Biochem. Physiol. 74A: 817-822. Withers. P.C 1978. Models of diffusion-mediated gas exchange i n animal burrows. Am. Na t u r a l i s t . 112(988): 1101-1112. Zar, J.H. 1974. B i o s t a t i s t i c a l Analysis. Englewood C l i f f s , N.J. Prentice - H a l l . 620pp. 132 APPENDIX I. Mean concentrations of test gas mixtures. A l l values are mean 1 standard error for fractional gas composition (X). TEST GAS S. late r a l i s S. columbianus MIXTURE Awake Hiber- Awake Awake Periodic Hiber-22°C nating 22°C 5°C Arousal 5C nating 15X02 15.14 15.21 15.08 15.08 15.04 15.3 •0.04 •0.08 •0.05 •0.05 ±0.01 10.11 10X02 10.14 10.22 10.12 10.16 10.22 10.13 ±0.05 •0.04 ±.0.05 10.03 10.03 10.06 5X02 4.96 5.04 5.10 5.17 5.33 5.10 ±0.13 ±0.10 •0.04 ±0.05 10.17 10.04 3X02 3.33 3.14 3.06 - 3.10 3.14 ±.0.03 ±.0.08 ±.0.03 •0.06 10.05 2XC02 1.96 2.08 1.88 2.05 2.13 2.05 ±0.11 •0.04 ±0.21 ±0.10 ±0.02 10.03 3XC02 2.95 3.07 3.07 3.13 3.02 3.12 •0.09 ±0.04 •0.03 •0.08 ±0.04 10.08 5XC02 5.17 5.16 5.12 5.26 5.12 5.09 •0.18 ±0.06 •0.05 ±.0.14 ±.0.06 10.04 7XC02 7.08 6.99 7.08 7.21 7.03 7.11 ±0.11 •0.12 •0.13 ±0.07 •0.03 10.07 15X02 15.16 15.16 15.10 15.19 15.23 15.06 3XC02 ±0.08 • 0.06 ±0.05 • 0.07 •0.13 10.07 3.00 3.11 3.11 3.11 3.07 3.02 ±0.13 ±0.08 ±0.17 ±0.10 ±0.05 10.10 15X02 15.09 15.24 15.12 15.26 15.24 15.11 5XC02 ±0.09 ±0.08 ±0.06 ±.0.07 10.16 10.06 4.96 5.18 4.91 5.07 5.07 5.08 •0.09 ±0.08 ±0.20 •0.11 10.04 10.04 10X02 10.15 10.19 10.19 10.24 10.27 10.15 3XC02 ±.0.11 ±0.08 ±0.13 ±0.13 10.12 10.06 3.13 3.04 3.17 3.17 3.02 3.02 ±.0.06 •0.07 •0.14 ±.0.09 10.02 10.06 10X02 10.36 10.26 10.27 10.41 10.12 10.10 5XC02 •0.09 ±0.07 • 0.13 •0.03 10.07 10.06 5.07 5.16 5.22 5.00 5.08 4.99 •0.06 ±0.07 •0.16 ±.0.09 10.09 l 0 . l l 50X02 51.1 50.4 50.5 51.9 49.7 50.7 ±0.46 • 0.10 •0.56 ±0.71 10.62 10.48 50X02 50.5 49.1 48.6 49.0 48.2 49.8 3XC02 ±.1.37 ±.0.33 •0.68 •0.43 10.97 10.96 3.15 3.12 3.11 3.13 3.21 3.10 ±.0.12 ±0.26 ±.0.10 ±.0.09 10.05 10.07 50X02 49.7 49.4 48.97 47.4 49.5 49.7 5XC02 •1.64 • 0.26 •0.36 ±0.80 10.79 10.61 5.13 5.11 4.98 5.14 5.15 5.14 •0.08 •0.05 •0.14 •0.08 •0.08 •0.08 133 

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